ES2592216T3 - Human anti-PD-1, PD-L1 and PD-L2 antibodies and their uses - Google Patents

Abstract

An isolated antibody or an antigen binding fragment thereof, comprising: a) a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 34-38; and b) a light chain variable region sequence selected from the group consisting of SEQ ID NO: 39-42, wherein the isolated antibody, or an antigen-binding fragment thereof, binds to a PD-L1 protein that it has the amino acid sequence of SEQ ID NO: 4, and the isolated antibody, or the antigen-binding fragment thereof, is humanized or composed.

Description

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DESCRIPTION

Human anti-PD-1, PD-L1 and PD-L2 antibodies and their uses Background of the invention

For T lymphocytes to respond to exogenous polypeptides, at least two signals must be provided through antigen presenting cells (APC) to inactive T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med. 165 : 302-319; Mueller, DL et al. (1990) J. Immunol. 144: 3701-3709). The first signal, which confers specificity to the immune response, is transduced by the T-cell receptor (TCR) after recognition of the exogenous antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, called costimulation, induces T lymphocytes to proliferate and become functional (Lenschow et al. (1996) Annu. Rev. Immunol. 14: 233). Costimulation is not antigen specific, nor limited to MHC, and is provided by different cell surface molecules expressed by APCs (Jenkins, MK et al. (1988) J. Immunol. 140: 3324-3330; Linsley, PS et al. (1991) J. Exp. Med 173: 721-730; Gimmi, CD et al. (1991) Proc. Natl. Acad Sci. United States 88: 6575-6579; Young, JW et al. (1992) J Clin. Invest. 90: 229-237; Koulova, L. et al. (1991) J. Exp. Med. 173: 759-762; Reiser, H. et al. (1992) Proc. Natl. Acad Sci. United States 89: 271-275; van-Seventer, GA et al. (1990) J. Immunol. 144: 4579-4586; LaSalle, JM et al. (1991) J. Immunol. 147: 774-80; Dustin, MI et al. (1989) J. Exp. Med. 169: 503; Armitage, RJ et al. (1992) Nature 357: 80-82; Liu, Y. et al. (1992) J. Exp. Med 175: 437-445).

Proteins B7-1 (CD80) and B7-2 (CD86) are critical costimulatory molecules (Freeman et al. (1991) J. Exp. Med. 174: 625; Freeman et al. (1989) J. Immunol. 143: 2714; Azuma et al. (1993) Nature 366: 76; Freeman et al. (1993) Science 262: 909). B7-2 plays a predominant role during primary immune responses, while B7-1, which is positively regulated later during an immune response, may be important in prolonging primary T lymphocyte responses or co-stimulating secondary T lymphocyte responses (Bluestone (1995) Immunity 2: 555).

CD28 is a ligand for B7-1 and B7-2 that is constitutively expressed in inactive T lymphocytes and increases in expression after activation of T lymphocytes. The binding of CD28 together with a TCR signal results in the transduction of a signal. costimulator that induces T lymphocytes to proliferate and secrete IL-2 (Linsley, PS et al. (1991) J. Exp. Med. 173: 721-730; Gimmi, CD et al. (1991) Proc. Natl. Acad Sci. United States 88: 6575-6579; June, CH et al. (1990) Immunol. Today 11: 211-6; Harding, FA et al. (1992) Nature 356: 607-609). A second ligand B7-1 and B7-2, CTLA4 (CD152), is homologous to CD28, but is not expressed in inactive T lymphocytes. The expression of CTLA4 occurs after T lymphocyte activation (Brunet, J. F. et al. (1987) Nature 328: 267270). Ligation of CTLA4 results in the transduction of an inhibitory signal that prevents T lymphocyte proliferation and cytokine secretion. Therefore, CTLA4 is a critical negative regulator of T lymphocyte responses (Waterhouse et al. (1995) Science 270: 985) (Allison and Krummel (1995) Science 270: 932). The third member of the CD28 family discovered is ICOS (Hutloff et al. (1999) Nature 397: 263; WO 98/38216). Linking ICOS with its ligand (ICOS-L) produces high levels of cytokine expression, but limited expansion of T lymphocytes (Riley JL et al. (2001) J. Immunol. 166: 4943-48; Aicher A. et al . (2000) J. Immunol. 164: 4689-96; Mages HW et al. (2000) Eur. J Immunol. 30: 1040-7; Brodie D. et al. (2000) Curr. Biol. 10: 333- 6; Ling V. et al. (2000) J. Immunol. 164: 1653-7; Yoshinaga SK et al. (1999) Nature 402: 827-32). If T lymphocytes are stimulated through the T lymphocyte receptor in the absence of a costimulatory signal, they become numb, energetic or die.

The importance of the costimulatory route B7: CD28 / CTLA4 / ICOS has been demonstrated in vitro and in various in vivo model systems. Blocking this costimulatory pathway produces the development of specific antigen tolerance in murine and human systems (Harding, FA et al. (1992) Nature 356: 607 609; Lenschow, DJ et al. (1992) Science 257: 789,792; Turka, LA et al. (1992) Proc. Natl. Acad. Sci. USA 89: 11102 11105; Gimmi, CD et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6586 6590; Boussiotis, V. et al. (1993) J. Exp. Med. 178: 1753 1763). Conversely, the expression of B7 by negative B7 murine tumor cells induces specific immunity mediated by T lymphocytes accompanied by tumor rejection and prolonged protection against tumor exposure (Chen, L. et al. (1992) Cell 71: 1093 1102; Townsend, SE and Allison, JP (1993) Science 259: 368 370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci. 90: 5687 5690.). Therefore, manipulation of costimulatory pathways offers great potential to stimulate or suppress immune responses in humans.

The discovery of more members of families B7-1 and CD28 has revealed additional routes that provide secondary costimulatory and inhibitory signals to T lymphocytes. One of the most novel routes is represented by the programmed death receptor 1 (PD-1; also called CD279) and its ligands, PD-L1 (B7-H1; CD274) and PD-l2 (B7-DC; CD273). PD-1 is a member of the CD28 / CtLa4 family that is expressed in activated T lymphocytes, but not at rest (Nishimura et al. (1996) Int. Immunol. 8: 773). The binding of PD-1 through its ligands mediates an inhibitory signal that results in reduced cytokine production, and reduced survival of T lymphocytes (Nishimura et al. (1999) Immunity 11: 141; Nishimura et al. (2001) Science 291: 319; Chemnitz et al. (2004) J. Immunol. 173: 945).

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PD-L1 is a member of the B7 family that is expressed in many types of cells, including APC and activated T lymphocytes (Yamazaki et al. (2002) J. Immunol. 169: 5538). PD-L1 is used for both PD-1I and B7-1. Binding of both B7-1 by PD-L1 expressed in T lymphocytes and binding of PD-L1 expressed in T lymphocytes by B7-1 results in inhibition of T lymphocytes (Butte et al. (2007) Immunity 27: 111 ). There is also evidence that, like other members of the B7 family, PD-L1 can also provide costimulatory signals to T lymphocytes (Subudhi et al. (2004) J. Clin. Invest. 113: 694; Tamura et al. ( 2001) Blood 97: 1809). WO 2008/083174 shows a method of reactivation of depleted T lymphocytes and for treating infections and tumors with a blocking murine anti-PD-L1 monoclonal antibody.

PD-L2 is a member of the B7 family expressed in various APCs, including dendritic cells, macrophages and mast cell-derived mast cells (Zhong et al. (2007) Eur. J. Immunol. 37: 2405). PD-L2 expressed in APC can inhibit both T lymphocyte activation through PD-1 ligation and co-stimulate T lymphocyte activation, through an independent PD-1 mechanism (Shin et al. (2005) J. Exp. Med. 201: 1531). In addition, the binding of PD-L2 expressed in dendritic cells produces an expression and survival of cytokines in enhanced dendritic cells (Radhakrishnan et al. (2003) J. Immunol. 37: 1827; Nguyen et al. (2002) J. Exp Med. 196: 1393). The structure and expression of PD-1, PD-L1 and PD-L2, as well as characteristics of signaling and functions of these molecules in the context of regulation of activation and tolerance of T lymphocytes (for example, therapeutic effects) is reviewed with more details in Kier et al. (2008) Ann. Rev. Immunol. 26: 677. The manipulation of this and other costimulatory pathways offers great potential to stimulate or suppress immune responses in humans and there is a need for compositions and methods that are useful for performing such manipulations.

Summary of the invention

The present invention is based on the generation and isolation of new compounds, human monoclonal antibodies as defined in the claims, which specifically bind human PD-L1

as well as the characterization of said new antibodies and the demonstration of their therapeutic value in the

treatment of various conditions mediated by PD-L1.

The usual techniques used to humanize murine antibodies frequently produce humanized antibodies that have reduced antigen binding affinities compared to the original murine antibodies (Almagro and Fransson (2008) Frontiers in Bioscience 13: 1619-1633; Foote and Winter (1992) J. Mol. Biol. 224: 487-499; Hwang et al. (2005) Methods 36: 35-42). Surprisingly, it has been observed that the compound human antibodies of the present invention bind PD-L1 with affinities very close to those of murine antibodies. Additionally, conventional humanization techniques produce humanized antibodies that retain some murine sequence. As a result, said antibodies can retain immunogenicity when administered to humans. For example, the humanized CAMPATH® antibody elicits immunogenicity in approximately 50% of patients. The compound human antibodies of the present invention, on the other hand, come entirely from sequences of human origin. Therefore, they are possibly less significantly immunogenic and more therapeutically effective and useful when administered to human patients rather than human anti-PD-L1 antibodies. Accordingly, the human antibodies composed of the present invention provide an improved means for the treatment and prevention of disorders mediated by PD-L1, partly attributable to their unique specificity, affinity, structure, functional activity and the fact that they proceed of human antibody sequences. The present invention is also based on the discovery of new therapeutic applications for these antibodies, including their use in the treatment of infections and cancers by administering the compound human antibodies described herein.

This document describes an isolated antibody, or an antigen binding fragment thereof, that binds to a PD-1 protein, a PD-L1 protein or a PD-L2 protein (such as the PD-1 protein, PD-L1 or human PD-L2), in which the isolated antibody, or antigen binding fragment thereof, is chimeric, humanized, compound, human or human, and comprising one, two, three, four, five or six CDR sequences selected from the group consisting of SEQ ID NO: 7-24.

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-1 protein (such as a PD-1 protein comprising the amino acid sequence of SEQ ID NO: 2), in which The isolated antibody, or antigenic binding fragment thereof, is chimeric, humanized, compound, human or human and comprising a heavy chain variable region sequence comprising SEQ ID NO: 7-9 (CDR1 sequence of SEQ ID NO : 7, CDR2 sequence of SEQ ID NO: 8, and CDR3 sequence of SEQ ID NO: 9) and / or a light chain variable region sequence comprising SEQ ID NO: 10-12 (CDR1 sequence of SEQ ID NO : 10, CDR2 sequence of SEQ ID NO: 11 and CDR3 sequence of SEQ ID NO: 12).

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-L1 protein (such as a PD-L1 protein comprising the amino acid sequence of SEQ ID NO: 4), in which the isolated antibody, or antigenic binding fragment thereof, is chimeric, humanized, compound,

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human or human, and comprising a heavy chain variable region sequence comprising SEQ ID NO: 13-15 (CDR1 sequence of SEQ ID NO: 13, CDR2 sequence of SEQ ID NO: 14, and CDR3 sequence of SEQ ID NO: 15), and / or a light chain variable region sequence comprising SEQ ID NO: 16-18 (CDR1 sequence of SEQ ID NO: 16, CDR2 sequence of SEQ ID NO: 17 and CdR3 sequence of SEQ ID NO: 18).

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-L2 protein (such as a PD-L2 protein comprising an amino acid sequence of SEQ ID NO: 6), in which the Isolated antibody, or antigen binding fragment thereof, is chimeric, humanized, compound, human or human, and comprising a heavy chain variable region sequence comprising SEQ ID NO: 19-21 (CDr1 sequence of SEQ ID NO : 19, CDR2 sequence of SEQ ID NO: 20 and CDR3 sequence of SEQ ID NO: 21), and / or a light chain variable region sequence comprising SEQ ID NO: 22-24 (CDR1 sequence of SEQ ID NO : 22, CDR2 sequence of SEQ ID NO: 23 and CdR3 sequence of SEQ ID NO: 24).

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-1 protein, a PD-L1 protein or a PD-L2 protein (such as a PD-1, PD-L1 protein or a protein) is also disclosed. Human PD-L2) in which the isolated antibody, or antigenic binding fragment thereof, is chimeric, humanized, compound and / or human, and comprising a heavy chain sequence selected from the group consisting of sEq ID NO: 25 -29, 34-38 or 43-47 or a sequence having a homology of at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more identical to the SEQ ID NO: 25-29, 34-38 or 43-47, and / or a light chain sequence selected from the group consisting of SEQ ID NO: 30-33, 39-42 or 48-51, or a sequence that It has a homology of at least approximately 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more with SEQ ID NO: 30-33, 39-42 or 48-51 .

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-1 protein, comprising the amino acid sequence of SEQ ID NO: 2, wherein the isolated antibody, or binding fragment is also disclosed. antigenic thereof, is chimeric, humanized, compound or human, and comprising a heavy chain sequence selected from the group consisting of SEQ ID NO: 25-29, or a sequence with an identity or homology of at least about 95% , 96%, 97%, 98%, 99%, 99.5%, 99.9% or more with SEQ ID NO: 25-29, and / or a light chain sequence selected from the group consisting of SEQ ID NO : 30-33, or a sequence with an identity or homology of at least about 95%, 96%, 97%, 98%, 99%,

99.5%, 99.9% or more with SEQ ID NO: 30-33. For example, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence of SEQ ID NO: 27 or 28, and a light chain variable region sequence of SEQ ID NO: 32 or 33. In some embodiments, the antibody or antigen binding fragment thereof described herein comprises a heavy chain variable region sequence of SEQ ID NO: 28, and a light chain variable region sequence of SEQ ID NO: 32.

The invention provides an isolated antibody, or an antigenic fragment thereof, that binds to a PD-L1 protein comprising the amino acid sequence of SEQ ID NO: 4, wherein the isolated antibody, or antigenic binding fragment thereof. , is humanized or composed and comprising a heavy chain sequence selected from the group consisting of SEQ ID NO: 34-38, and a light chain sequence selected from the group consisting of SEq ID NO: 39-42. For example, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence of SEQ ID NO: 35 or 37, and a light chain variable region sequence of SEQ ID NO: 39, 40 or 42. In some embodiments, the antigen binding antibody or fragment thereof comprises a heavy chain variable region sequence of SEQ ID NO: 35, and a light chain variable region sequence of SEQ ID NO: 42.

An isolated antibody, or an antigen binding fragment thereof, that binds to a PD-L2 protein comprising the amino acid sequence SEQ ID NO: 6, in which the isolated antibody, or antigen binding fragment of the It is also chimeric, humanized, compound or human and comprising a heavy chain sequence selected from the group consisting of SEQ ID NO: 43-47, a sequence with an identity or homology of at least about 95%, 96%, 97 %, 98%, 99%, 99.5%, 99.9% or more with SEQ ID NO: 43-47, and / or a light chain sequence selected from the group consisting of SEQ ID NO: 4851, or a sequence with an identity or homology of at least about 95%, 96%, 97%, 98%, 99%,

99.5%, 99.9% or more with SEQ ID NO: 48-51. For example, the antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence of SEQ ID NO: 44 or 46, and a light chain variable region sequence of SEQ ID NO: 49, 50 or 51. In some embodiments, the antigen binding antibody or fragment thereof comprises a heavy chain variable region sequence of SEQ ID NO: 46, and a light chain variable region sequence of SEQ ID NO: 51.

Another embodiment of the invention is an isolated antibody, or an antigen binding fragment thereof, that binds to a PD-L1 protein, in which the isolated antibody inhibits the binding of biotinylated 29E2A3 antibody to Fc-PD-L1 in a competition ELISA assay, wherein the 29E2A3 antibody comprises a heavy chain variable region sequence comprising SEQ ID NO: 78, and a light chain variable region sequence comprising SEQ ID no: 79.

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Another embodiment of the invention is an isolated antibody, or an antigen binding fragment thereof, that binds to a PD-L1 protein in which the isolated antibody inhibits the signal mediated by PD-L1.

In particular, one embodiment of the invention is an isolated nucleic acid as defined in the claims. Another embodiment is a vector, a host cell or an animal as defined in the claims.

Also disclosed is a nucleic acid that hybridizes, under stringent conditions, with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of SEQ ID NO: 25-51, or a sequence with a homology of at least about 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more with SEQ ID No: 25-51.

Thus, the invention provides an isolated nucleic acid as defined in the claims which encodes a heavy chain variable region and a light chain variable region of any of the antibodies or antigen binding fragments thereof of the invention. In some embodiments, the nucleic acid is a vector, such as an expression vector. The invention also provides a host cell comprising one or more nucleic acids encoding the heavy and light chain of the antibodies or antigen binding fragments described herein. In some embodiments, the host cell produces the antibodies or antigenic binding fragments. The invention also provides methods of producing the antibody or antigen binding fragment described herein which comprise culturing a cell that produces the antigen binding antibody or fragment and recovering the antibody or antigenic binding fragment from the cell culture.

Additionally, the invention includes a pharmaceutical composition as defined in the claims comprising an isolated antibody or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier.

The invention includes a method of reactivation of an exhausted T lymphocyte, which comprises contacting a population of T lymphocytes in which some cells express PD-L1 using an antibody described herein or an antigen binding fragment thereof well in vitro or ex vivo.

The invention further includes an antibody, or an antigen binding fragment thereof, as defined in the claims, for use in a method of treating a subject suffering from a persistent infection, including a viral infection, a bacterial infection, a helmintic infection or a protozoal infection by administering to the subject a composition comprising an effective amount of an isolated antibody described herein, or an antigen binding fragment thereof.

The invention further includes an antibody, or an antigen binding fragment thereof, as defined in the claims, for use in a method of treating a subject suffering from cancer by administering to the subject a composition comprising an effective amount of an antibody. isolated described herein, or an antigen binding fragment thereof, including in which the isolated antibody induces antibody-mediated cytotoxicity or is modified to induce antibody-mediated cytotoxicity or conjugate to an agent selected from the group consisting of a toxin or an image forming agent. The antibody or antigen-binding fragment that binds to PD-L1 is administered to the subject who has a cancer that overexpresses PD-L1.

A method of treating the subject suffering from asthma is also disclosed, which comprises administering to the subject a composition comprising an effective amount of an isolated antibody that binds to a PD-L2 protein described herein, or an antigenic binding fragment. of the same.

A method of treating a subject suffering from an inflammatory disease or transplant rejection is also disclosed, administering to the subject a composition comprising an effective amount of an isolated antibody described herein, or an antigenic binding fragment thereof, which bind to a PD-L1 protein.

Specifically, as defined in the claims, the invention provides an antibody, an antigen binding fragment or a polypeptide of the invention for use in methods of treating said disorders. The invention also includes the use of an antibody, an antigen binding fragment or a polypeptide described herein for the manufacture of a medicament, such as a medicament for the treatment of any of the diseases described herein in a subject. .

Brief description of the drawings

Figure 1 shows a schematic diagram of expression vectors used for cloning of assembled human immunoglobulin sequences of the present invention.

Figures 2A-2E show human heavy chain variable region sequences, composed (Figure 2A, VH1; Figure 2B, VH2; Figure 2C, VH3; and Figure 2D, VH4; Figure 2E, VH5) designed to correspond to those of the

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mouse anti-human PD-1 antibody, EH12.2H7.

Figures 3A-3D show of human light chain variable region sequences, composed (Figure 3A, Vk1; Figure 3B, Vk2; Figure 3C, Vk3; Figure 3D, Vk4) designed to correspond to that of the anti-PD-1 antibody Human mouse, EH12.2H7.

Figures 4A-4E show human heavy chain variable region sequences, composed (Figure 4A, VH1; Figure 4B, VH2; Figure 4C, VH3; Figure 4D, VH4; Figure 4E, VH5) designed to correspond to that of the anti-antibody -PD-LI human mouse, 29E.2A3.

Figures 5A-5D show human light chain variable region sequences, composed (Figure 5A, Vk1; Figure 5B, Vk2; Figure 5C, Vk3; Figure 5D, Vk4) designed to correspond to that of the human anti-PD-LI antibody of mouse, 29E.2A3.

Figures 6A-6E show human heavy chain variable region sequences, composed (Figure 6A, VH1; Figure 6B, VH2; Figure 6C, VH3; Figure 6D, VH4; Figure 6E, VH5) designed to correspond to that of the anti-antibody -PD-L2 human mouse, 24F.10C12.

Figures 7A-7D show sequences of the human light chain variable region, composed (Figure 7A, Vk1; Figure 7B, Vk2; Figure 7C, Vk3; Figure 7D, Vk4) designed to correspond to that of the anti-PD-L2 antibody mouse human, 24F.10C12.

Figures 8A-8C show SDS-PAGE results of 1 | jg of human antibodies, compounds, corresponding to mouse anti-human antibodies, EH12.2H7, 29E.2A3 and 24F.10C12, respectively.

Figure 9A-9C shows ELISA competition results of human antibodies corresponding to and with respect to mouse anti-human antibodies, EH12.2H7, 29E.2A3 and 24F.10C12, respectively. In Figure 9A, the binding of purified antibodies with human PD-1 were assayed by competition ELISA. Various concentrations of each antibody (0.06 jg / ml at 8 jg / ml) were mixed with a fixed concentration of biotinylated EH12.2H7 (40 ng / ml) and bound with a maxisorb immulon plate coated with PD-1. The binding was also detected by streptavidin-HRP and OPD substrate. The absorbance at 490 nm was measured on a plate reader and this was plotted against the concentration of the test antibody. In Figure 9B, the binding of the purified antibodies with human PD-L1 was assayed by competition ELISA. Different concentrations of each antibody (0.02 mg / ml to 8 mg / ml) were mixed with a fixed concentration of biotinylated 29E.2A3 (40 ng / ml) and bound to a maxisorb immulon plate coated with PD-L1. Binding was detected with streptavidin-HRP and OPD substrate. The absorbance at 490 nm was measured on a plate reader and this was plotted against the concentration of the test antibody. In Figure 9C, the binding of purified antibodies against human PD-L2 was assayed by competition ELISA. Various concentrations of each antibody (0.02 mg / ml to 8 mg / ml) were mixed with a fixed concentration of biotinylated 24F.10C12 (40 ng / ml) and bound with a maxisorb immulon plate coated with PD-L2. Binding was detected by streptavidin-HRP and OPD substrate. The absorbance at 490 nm was measured on a plate reader and this was plotted against the concentration of the test antibody.

Figures 10A-10C show IC50 binding data resulting from ELISA competition analysis of human antibodies, compounds, formed according to different combinations of human heavy and light chains, composed, designed to correspond to those of anti-human antibodies. of mouse, EH12.2H7 (figure 10A), 29E.2A3 (figure 10b) and 24F.10C12 (figure 10C), respectively. The assay was performed as described in Figure 3. The IC50 of each heavy and light chain combination was normalized against the IC50 of the mouse antibody. SD = No data.

Figure 11 shows the amino acid sequences of PD-1, PD-L1 and PD-L2.

Figure 12 shows the amino acid sequences of the CDR regions of some of the human antibodies, compounds, described herein.

Figure 13 shows the amino acid sequences of the variable regions of some of the human antibodies, compounds, described herein.

Figures 14A and 14B show the effect of a humanized anti-PD-1 antibody and a humanized anti-PD-L1 antibody on the proliferative capacity of Gag-specific CD8 T lymphocytes of IVS in vitro. Each symbol represents an individual macaque. Numbers in parentheses represent an increase factor in proliferation in the presence of a blocking Ab compared to a non-blocking Ab.

Figure 15 shows that the PD-L1 block restores the proliferation driven by the intrahepatic CD8 T lymphocyte antigen (representative data of the animal 1564).

Detailed description of the invention

The present invention provides new therapeutic compounds based on antibodies for the treatment and diagnosis of various disorders mediated by PD-L1, (eg, treatment of persistent and cancerous infectious diseases).

So that the present invention can be more easily understood, first, certain terms are defined. Additional definitions are set forth throughout the detailed description.

As used herein, the terms "PD-1", "PD-L1" and "PD-L2" include any of the variants or isoforms that naturally express cells, and / or their fragments having the same less a biological activity of the full length polypeptide, unless expressly defined otherwise. In addition, the term "PD-1 ligand" includes any or both PD-L1 (Freeman et al. (2000) J. Exp. Med. 192: 1027) and PD-

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L2 (Latchman et al. (2001) Nat. Immunol. 2: 261) and any of the variants or isoforms that naturally express the cells, and / or their fragments that have at least one biological activity of the full-length polypeptides . For example, the sequences of PD-1, PD-L1 and PD-L2 of different species, including humans, are well known in the art (see, for example, Honjo et al., US Pat. 5,629,204, which discloses human and mouse PD-1 sequences; Wood et al., U.S. Patent No. 7,105,328, which discloses human PD-1 sequences; Chen et al., U.S. Pat. No. 6,803,192, which discloses human and mouse PD-L1 sequences; Wood et al., U.S. Patent No. 7,105,328, which discloses human PD-L1 sequences; Freeman et al., U.S. Patent United Pub. 20020164600, which reveals human and mouse PD-L2 sequences).

As used herein, the term "antibody" includes whole antibodies and any antigenic binding fragment (ie, "antigenic binding part") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy chains (H, heavy) and two light chains (L, light) interconnected by disulfide bonds, or an antigenic binding part thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a constant heavy chain region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a variable light chain region (abbreviated herein as Vl) and a constant light chain region. The light chain constant region comprises a domain, CL. The Vh and Vl regions can also be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called conserved framework regions (FR, framework regions). Each Vh and Vl comprises three CDR and four FR, arranged from the amino end to the carboxyl end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, fR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. "Inactivating antibodies" refers to antibodies that do not induce the complement system.

The term "hypervariable regions", "HVR" or "HV", when used herein refers to regions of an antibody variable domain that are sequence-variable and / or form structurally defined loops. six HVR; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 have the greatest diversity of the six HVR, and it is thought that H3 in particular function plays an exclusive role in conferring refined specificity to antibodies See, for example, Xu et al. Immunity 13: 37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248: 1-25 (Lo, ed., Human Press, Totowa, NJ, 2003)) In fact, naturally occurring camelid antibodies consisting of only one heavy chain are functional and stable in the absence of a light chain, see, for example, Hamers-Casterman et al., Nature 363: 446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3: 733-736 (1996).

Various delineations of hypervariable regions are in use and are included in this document. The determining regions of complementarity (CDR) of Kabat are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda , MD. (1991)). On the other hand, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 (see below) depending on the length of the loop (this is because the Kabat numbering scheme places the inserts in H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and both 35A and 35B are present, the loop ends at 34). AbM hypervariable regions represent a compromise between Kabat CDRs and Chothia structural loops, and are used in the AbM Oxford Molecular antibody modeling software. Hypervariable regions of "contact" are based on an analysis of the complex crystalline structures available. The remains of each of these hypervariable regions are indicated below.

 Core

 Kabat AbM Chothia Contact

 L1

 L24-L34 L24-L34 L24-L34 L30-L36

 L2

 L50-L56 L50-L56 L50-L56 L46-L55

 L3

 L89-L97 L89-L97 L89-L97 L89-L96 H30-H35B (numbering of

 H1

 H31-H35B H26-H35B H26-H32, 33 or 34 Kabat)

 H1

 H31-H35 H26-H35 H26-H32 H30-H35 Chothia) (numbering of

 H2

 H50-H65 H50-H58 H52-H56 H47-H58

 H3

 H95-H102 H95-H102 H95-H102 H93-H101

The hypervariable regions may comprise the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35B (H1) , 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. These extended hypervariable regions are normally combinations of the definitions of Kabat and

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Chothia, which may optionally also include remains identified using the definition of Contact. The remainders of the variable domain are listed according to Kabat et al., Cited above for each of these definitions.

The remains of the "conserved frame" or "FR" region are those variable domain residues other than the HVR residues as defined herein.

The expression "numeration of variable domain residues as in Kabat" or "amino acid position numeration as in Kabat", and variations thereof, refers to the numbering system used for heavy chain variable domains or variable chain domains light of the set of antibodies in Kabat et al., cited above. Using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids corresponding to a shortening of, or insertion into, an FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and residues inserted (eg, residues 82a, 82b and 82c, etc. according to Kabat ) after rest 82 of the heavy chain FR. The numbering of Kabat residues can be determined for an antibody determined by alignment in regions of homology of the antibody sequence with a "conventional" numbered Kabat sequence.

The term "region Fc" herein is used to define a terminal C region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the limits of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of human IgG heavy chain is usually defined to range from an amino acid residue at position Cys226, or from Pro230, to the carboxyl end thereof. . The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during the production or purification of the antibody, or by recombinant modification of nucleic acid genetic engineering encoding a heavy chain of the antibody. Accordingly, an intact antibody composition may comprise antibody populations with all K447 residues removed, antibody populations without K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies of the invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

"The Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII and FcyRIII subclasses including allelic variants and alternative splicing forms of these receptors, FcyRII receptors include FcyRNA ( an "activator receptor") and FcyRIIB (an "inhibitor receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. The FcyRNA activator receptor contains a tyrosine-based immunoreceptor activation motif (ITAM). ) in its cytoplasmic domain The FcyRIIB inhibitor receptor contains a reason for tyrosine-based immunoreceptor inhibition (ITIM) in its cytoplasmic domain (see M. Daeron, Annu. Rev. Immunol. 15: 203-234 (1997). FcR in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med 126: 330-41 (1995) Other FcRs that include those who will identify in the future are encompassed by the term "FcR" in this document.

The term "CDR" refers to a complementarity determining region (CDR) of which three constitute the union character of a variable light chain region (CDRL1, CDRL2 and CDRL3) and three constitute the union character of a region heavy chain variable (CDRH1, CDRH2 and CDRH3). The CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences comprising conserved framework or framework regions. The CDR limits of exact definitions and lengths are subject to different classification and numbering systems. Therefore, the CDRs can refer to those of Kabat, Chothia and Contact or any other limit definition, including the numbering system described in this document. Despite different limits, each of these systems has some degree of overlap in what constitute the so-called "hypervariable regions", within the variable sequences. The CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent conserved framework region. See, for example, Kabat, Chothia and / or MacCallum et al., (Kabat et al., In "Sequences of Proteins of Immunological Interest," 5th Edition, US Department of Health and Human Services, 1992; Chothia et al., J. Mol. Biol., 1987, 196: 901; and MacCallum et al., J. Mol. Biol., 1996, 262: 732).

As used herein, the term "antigen binding part" of an antibody (or simply "antigenic part"), refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (by example, PD-1, PD-L1 and / or PD-L2). It has been observed that the antigen binding function of an antibody can be carried out by fragments of a full length antibody. Examples of binding fragments included within the expression "antigen binding part" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vh, Vl, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of domains Vh and CH1; (iv) an Fv fragment consisting of the single arm Vh and Vl domains of the antibody; (v) a dAb fragment (Ward et al., (1989) Nature 341: 544 546), which consists of a Vii domain; (vi) a region determining complementarity (CDR)

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isolated or (vii) a combination of two or more isolated CDRs that can optionally be joined by a synthetic linker. Additionally, although the two domains of the Fv fragment, Vh and Vl, are encoded by different genes, they can be linked, using recombinant methods, by a synthetic linker that allows them to be constituted as a simple protein chain in which the Vh and Vl regions they mate to form monovalent molecules (known as single chain Fv (scFv); see for example Bird et al. (1988) Science 242: 423 426; and Huston et al. (1988) Proc. Natl. Acad. Sci. United States 85 : 5879 5883). Said single chain antibodies are also intended to be included within the expression "antigen binding part" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and the fragments are screened for their usefulness in the same manner as intact antibodies.

The antibodies can be polyclonal or monoclonal; xenogenic, allogenic or singenic; or modified forms thereof (for example, humanized, chimeric, etc.). The antibodies can also be completely human. Preferably, the antibodies of the invention specifically or substantially bind to PD-1, PD-L1 or PD-L2 polypeptides. The term "monoclonal antibody" as used herein, refers to an antibody that has a single specificity and binding affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody that has a single binding specificity and has variable and constant regions derived from human germline immunoglobulin sequences or from immunoglobulin that are not from the germ line. In one embodiment, the human monoclonal antibodies are produced by a hybridoma that includes a B lymphocyte obtained from a non-human transgenic animal, for example, a transgenic mouse, which has a genome comprising a human heavy chain transgene and a light chain transgene. fused to an immortalized cell.

As used herein, the term "isolated antibody" is intended to refer to an antibody that substantially lacks other antibodies that have different antigenic specificities (eg, an isolated antibody that specifically binds to PD-1, PD-L1 or PD-L2 substantially lacks antibodies that do not bind PD-1, PD-L1 or PD-L2, respectively). An isolated antibody that specifically binds to an epitope of PD-1, PD-L1 and / or PD-L2 may, however, have cross-reactivity with other PD-1, PD-L1 and / or PD-L2 proteins, respectively, of different species. However, the antibody always binds preferably to PD-1, PD-L1 and / or human PD-L2. In addition, the isolated antibody normally lacks substantially other cellular material and / or chemical compounds. A combination of "isolated" monoclonal antibodies having different specificities for PD-1, PD-L1 and / or PD-L2 combined in a well defined composition is disclosed herein.

As used herein, the term "humanized antibody" refers to an antibody consisting of the CDR of antibodies derived from non-human mammals, and the FR region and the constant region of a human antibody. A humanized antibody is useful as an effective component in a therapeutic agent according to the present invention since the antigenicity of the humanized antibody in the human body is reduced.

As used herein, the term "compound antibody" refers to an antibody that has variable regions comprising germline or non-germline immunoglobulin sequences of two or more unrelated variable regions. Additionally, the term "human, compound antibody" refers to an antibody having constant regions derived from human germline or non-germline immunoglobulin sequences and variable regions comprising human germline or non-germline sequences of two or more unrelated human variable regions. A human antibody, compound, is useful as an effective component in a therapeutic agent according to the present invention since the antigenicity of the human antibody, compound, in the organism is reduced.

As used herein, the term "recombinant human antibody" includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (eg, from a mouse ) which is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (further described in section I, below), (b) antibodies isolated from a transformed host cell to express the antibody, for example, from an antibody transfectome, (c) antibodies isolated from a library of recombinant human combinatorial antibodies and (d) antibodies prepared, expressed, created or isolated by any other means involving the splicing of human immunoglobulin gene sequences from other DNA sequences . Such recombinant human antibodies have variable and constant regions derived from human germline and non-germline immunoglobulin sequences. However, in certain embodiments, said recombinant human antibodies may undergo mutagenesis in vitro (or, when a transgenic animal is used for human Ig sequences, somatic mutagenesis in vivo) and therefore the amino acid sequences of the Vh and Vl regions of Recombinant antibodies are sequences that, although derived from and related to human germline sequences Vh and Vl, may not exist in nature in the germline repertoire of human antibodies in vivo.

As used herein the expression "heterologous antibody" is defined in relation to the transgenic non-human organism that produces said antibody. That term refers to an antibody that has a sequence

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of amino acids or to a nucleic acid sequence corresponding to that found in an organism that does not consist of the transgenic non-human animal and generally of a species other than that of the transgenic non-human animal.

As used herein, the term "Kd" is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction.

As used herein, the term "specific binding" refers to an antibody that binds to a predetermined antigen. Normally, the antibody binds with an affinity (Kd) of approximately less than 10-7 M, such as approximately 10-8 M, 10-9 M or 10-10 M or even lower when determined by resonance technology of Superficial plasmon (SPR) in a BIACORE 3000 instrument using recombinant human PD-1, PD-L1 or PD-L2 as the analyte and antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4 , 0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 times or greater than their affinity for binding with a non-specific antigen (for example, BSA, casein) other than the predetermined antigen or a closely related antigen. The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the expression "an antibody that specifically binds an antigen."

As used herein, the term "isotype" refers to the class of antibody (eg, IgM or IgG1) that is encoded by the heavy chain constant region genes.

As used herein, the term "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently bound to a protein, more specifically an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns that occur in nature in antibodies produced by the non-human transgenic animal species, when a person skilled in the art would recognize that the glycosylation pattern of the Heterologous antibody is more similar to said glycosylation pattern in the species of the non-human transgenic animal rather than the species from which the CH genes of the transgenic gene originate.

As used herein, the expression "natural origin" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is of natural origin.

As used herein, the term "rearranged" refers to a configuration of a heavy chain or light chain immunoglobulin locus in which a segment V is placed immediately adjacent to a DJ or J segment in a conformation encoding essentially a complete Vh and Vl domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison with the germline DNA; a rearranged locus will have at least one element of recombinant heptameric / nonameric homology.

As used herein, the expression "not rearranged" or "germline configuration" in reference to a segment V refers to the configuration in which segment V is not recombined such that it is immediately adjacent to a segment D or J.

As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. A nucleic acid molecule can be single or double stranded, but preferably it is double stranded DNA.

As used herein, the term "isolated nucleic acid molecule" in reference to nucleic acids encoding antibodies or antibody parts (eg, Vh, Vl, CDR3) that bind to PD-1, PD-L1 or PD-L2, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the antibody or part of the antibody lack other nucleotide sequences encoding antibodies or parts of antibodies that bind to antigens other than PD-1, PD-L1 or PD-L2, respectively, with other sequences that can naturally flank the nucleic acid in human genomic DNA. Figures 2-7 correspond to nucleotide and amino acid sequences comprising the heavy chain (Vh) and light chain (Vl) variable regions of the human anti-PD-1, PD-L1 or PD-L2 antibodies disclosed herein. document, respectively.

"Conservative sequence modifications" of the sequences set forth in the figures are also disclosed herein (eg, Figures 2-7), including modifications of nucleotide and amino acid sequences that do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include substitutions, additions and deletions of nucleotides and amino acids. Modifications can be made to the sequence set forth in the figures (for example, Figures 2-7) by conventional techniques known in the art, such as site-directed mutagenesis, and PCR-mediated mutagenesis. The

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Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acid side chains (aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine , cysteine, tryptophan), non-polar side chains (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched beta side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue predicted in an anti-PD-1, anti-PD-LI or human anti-PD-L2 antibody is preferably replaced with another amino acid residue of the same side chain family.

Alternatively, in another embodiment, random mutations may be introduced throughout all or part of a sequence encoding a human anti-PD-1, PD-L1 or PD-L2 antibody, such as by saturation mutagenesis, and Resulting modified human anti-PD-1, anti-PD-LI or anti-PD-L2 antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the heavy and light chain variable region nucleotide sequences disclosed herein and / or which contain the heavy and light chain variable region amino acid sequences disclosed herein (for example, Figures 2-7) include substantially similar antibodies encoded by or containing similar sequences that have been modified in a conservative manner. A further analysis of how such substantially similar antibodies can be generated based on the sequences (ie, heavy and light chain variable regions) disclosed herein (eg, Figures 2-7) are provided below.

Additionally, there is a known and defined correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can encode the protein, as defined in the genetic code (see below). Similarly, there is a known and definitive correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE

 Alanina (Ala, A)

 GCA, GCC, GCG, GCT

 Arginine (Arg, R)

 AGA, ACG, CGA, CGC, CGG, CGT

 Asparagine (Asn, N)

 AAC, AAT

 Aspartic Acid (Asp, D)

 GAC, GAT

 Cysteine (Cys, C)

 TGC, TGT

 Glutamic Acid (Glu, E) GAA, GAG

 Glutamine (Gln, Q)

 CAA, CAG

 Glycine (Gly, G)

 GGA, GGC, GGG i, GGT

 Histidine (His, H)

 CAC, CAT

 Isoleucine (Ile, I)

 ATA, ATC, ATT

 Leucine (Leu, L)

 CTA, CTC, CTG, CTT, TTA, TTG

 Lysine (Lys, K)

 AAA, AAG

 Methionine (Met, M)

 ATG

 Phenylalanine (Phe, F)

 TTC, TTT

 Proline (Pro, P)

 CCA, CCC, CCG, CCT

 Serina (Ser, S)

 AGC, AGT, TCA, TCC, TCG, TCT

 Threonine (Thr, T)

 ACA, ACC, ACG, ACT

 Tryptophan (Trp, W)

 TGG

 Tyrosine (Tyr, Y)

 TAC, TAT

 Valine (Val, V)

 GTA, GTC, GTG, GTT

 Termination sign

 (finalization)

 TAA, TAG, TGA

An important and well-known characteristic of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one triplet of coding nucleotides (illustrated above) can be used. Therefore, several different nucleotide sequences can encode a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms can translate some sequences more efficiently than others do). Additionally, occasionally, a methylated variant of a purine or pyrimidine may

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found in a particular nucleotide sequence. Such methadones do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared are identical, with appropriate nucleotide insertions or deletions, at least about 80 % of the nucleotides, usually at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more of the nucleotides, and more preferably at least about 97%, 98%, 99 % or more of the nucleotides. Alternatively, there is substantial homology when the segments hybridize under conditions of selective hybridization with the complement of the chain.

The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (that is,% of identity = no. Of identical positions / total of no. Of positions x 100), taking into account the number of gaps and the length of each hole, which need to be introduced for optimal alignment of the two sequences. Sequence comparison and determination of percent identity between two sequences can be performed using a mathematical algorithm, as described in the non-limiting examples below.

The percentage of identity between two nucleotide sequences can be determined using the GAP program of the GCG software package (available online at the GCG company website), using a NWSgapdna.CMP matrix and a gap weight of 40, 50 , 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11 17 (1989)) that has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight wreckage table, a gap length penalty of 12 and a gap penalty of 4. In addition , the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. (48): 444 453 (1970)) that has been incorporated into the GAP program in the GCG software package ( available on the internet on the website of the company GCG), using either a ma triz Blosum 62 or a matrix PAM250, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1,2, 3, 4, 5 or 6.

The nucleic acid and protein sequences of the present invention can additionally be used as a "sequence to be investigated" to perform a search against public databases to identify, for example, related sequences. Such searches can be performed using the NBLAST and XBLAST (version 2.0) programs of Altschul, et al. (1990) J. Mol. Biol. 215: 403 10. BLAST nucleotide searches can be performed with the NBLAST program, punctuation = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid sequences of the invention. BLAST protein searches can be performed with the XBLAST program, punctuation = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain hollow alignments for comparative purposes, BLAST with gaps can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389 3402. When using BLAST and BLAST programs with gaps, You can use the default parameters of the respective programs (for example, XBLAST and NBLAST) (available online on the NCBI website).

Nucleic acids may be present in whole cells, in a cell lysate or in a pure partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when purified from other cellular components or other contaminants such as, for example, other nucleic acids or cellular proteins, by conventional techniques, including alkaline / SDS treatment, CsCl band, chromatography in column, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, although often in a native sequence (except for modified restriction sites and the like), of any cDNA, genomic or mixtures thereof can be mutated according to conventional techniques to provide genetic sequences. . For coding sequences, these mutations can affect the amino acid sequence if desired. In particular, DNA sequences substantially homologous to or derived from the V, D, J, native, and other constants described herein are contemplated (in which "derivative" indicates that a sequence is identical or modified from another sequence).

A nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcriptional regulatory sequences, operably linked means that the DNA sequences that are linked are contiguous and, when necessary, join two protein-coding regions, contiguous and in reading phase. For switching sequences, operably linked indicates that the sequences may be able to effect the switching recombination.

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As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid with which it has been linked. One type of vector is a "plasmid" that refers to a circular single-stranded DNA loop in which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional segments of DNA can be ligated into the viral genome. Certain vectors are capable of performing autonomous application in a host cell into which they are introduced (for example, bacterial vectors having an origin of bacterial replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell, and therefore replicated with the host genome. Additionally, certain vectors can direct the expression of genes with which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as plasmid in the most commonly used vector form. However, the invention aims to include such different forms of expression vectors, such as viral vectors (eg, retroviruses with replication defects, adenoviruses and adeno-associated viruses) that perform equivalent functions.

As used herein, the expression "recombinant host cell" (or simply "host cell"), is intended to refer to a cell in which a recombinant expression vector has been introduced. It should be understood that said terms are included to refer not only to the particular subject cell but to the offspring of said cell. Since certain modifications may occur in successive generations due to mutation or influences in the environment, such offspring may, in fact, not be beneficial for the parental cell, but would still be within the term "host cell" as used herein. document.

As used herein, the term "subject" includes any human or non-human animal. For example, the compositions of the present invention can be used to treat a subject with an inflammatory disease, such as arthritis, for example, rheumatoid arthritis. The term "non-human animal" includes all vertebrates, for example, mammals and non-mammals such as nonhuman primates, goat, dog, cow, chickens, amphibians, reptiles, etc.

As used herein, the term "modular" includes positive and negative regulation, for example, enhancing or inhibiting a response.

As used herein, the term "inhibit" includes the decrease, limitation or blocking of, for example, a particular action, function or interaction.

As used herein, the term "immune cell" refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B and T lymphocytes; natural cytolytic cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.

As used herein, the term "T lymphocyte" includes CD4 + T lymphocytes and CD8 + T lymphocytes. The expression T lymphocyte also includes auxiliary T type T lymphocytes 1, auxiliary T type T lymphocytes 2, auxiliary T type T lymphocytes 17 and inhibitor T lymphocytes. The expression "antigen presenting cell" includes professional antigen presenting cells (eg, B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (eg, keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes).

As used herein, the term "immune response" includes immune responses mediated by B lymphocytes and / or mediated by T lymphocytes that are influenced by modulation of costimulation of T lymphocytes. Immune responses by way of example include responses. of T lymphocytes, for example, cytokine production and cell cytotoxicity. In addition, the expression immune response includes immune responses that are indirectly affected by the activation of T lymphocytes, for example, production of antibodies (humoral responses) and activation of cytokine-sensitive cells, for example, macrophages.

As used herein, the term "costimulatory", as used with reference to activated immune cells includes the ability of a costimulatory polypeptide to provide a second signal mediated by a non-activating receptor (a "costimulatory signal") that induces proliferation and / or effector function. For example, a costimulatory signal may result in the secretion of cytokines, for example, in a T lymphocyte that has received a signal mediated by T-cell receptors. Immune cells that have received a signal mediated by cell receptors, for example. , by means of an activation receptor they are referred to herein as "activated immune cells".

As used herein, the term "inhibitory signal" refers to a signal transmitted by an inhibitor receptor (eg, CTLA4 or PD-1) for a polypeptide in an immune cell. Said signal

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it antagonizes a signal by an activation receptor (for example, by a TCR or CD3 polypeptide) and can result, for example, in the inhibition of a second generation of messengers; a proliferation inhibition; an inhibition of the effector function in the immune cell, for example, reduced phagocytosis, reduced antibody production, reduced cell cytotoxicity, the decrease of the immune cell to produce mediators (such as cytokines (for example, IL-2) and / or mediators of allergic responses); or the development of anergia.

As used herein, the term "insensitive" includes reactivity of immune cells to stimulation, for example, by means of an activation receptor or a cytokine, insensitivity may occur, for example, due to exposure to immunosuppressants or exposure to high Antigen dose As used herein, the term "anergy" or "tolerance" includes refractivity upon activation of receptor-mediated stimulation. Such refractivity is generally antigen specific and persists after exposure to tolerant antigen has ceased. For example, anergy in T lymphocytes (as opposed to insensitivity) is characterized by the absence of cytokine production, for example, IL-2. T lymphocyte anergy occurs when T lymphocytes are exposed to an antigen and receive an first signal (a signal mediated by CD-3 or T-cell receptor) in the absence of a second signal (a costimulatory signal). ones, the re-exposure of the cells to the same antigen (even if re-exposure occurs in the presence of a costimulatory polypeptide) results in the inability to produce cytokines and therefore the inability to proliferate. However, anergic T lymphocytes can proliferate if grown with cytokines (for example, IL-2). For example, the energy of T lymphocytes can also be observed by the absence of IL-2 production by T lymphocytes measured by ELISA or by proliferation assay using an indicator cell line. As an alternative, a generic indicator construction can be used. For example, allergic T lymphocytes cannot initiate transcription of the induced IL-2 gene or by a heterologous promoter under the control of the IL-2 gene enhancer in 5 'or by a multimer of the API sequence that could be found within the enhancer (Kang et al. (1992) Science 257: 1134).

As used herein, the term "activity" when used with respect to a polypeptide, for example, polypeptide PD-1, PD-L1 or PD-L2, includes activities that are inherent in the structure of the protein. For example, with respect to the PD-1 ligand, the term "activity" includes the ability to modulate costimulation of immune cells (for example, by modulating a costimulatory signal in an activated immune cell) or modulating inhibition by modulating an inhibitory signal in an immune cell (for example, by connecting a natural receptor to an immune cell). Those skilled in the art will recognize that when a PD-1 ligand polypeptide binds to a costimulatory receptor, a costimulatory signal can be generated in the immune cell. When a PD-1 ligand polypeptide binds to an inhibitor receptor, an inhibitory signal is generated in the immune cell. In addition, when a PD-1 ligand binds to a B7-1 polypeptide, an inhibitory signal can be generated (Butte et al. (2007) Immunity 27: 111).

With respect to PD-1, the term "activity" includes the ability of a PD-1 polypeptide to modulate an inhibitory signal in an immune cell, for example, by coupling a natural PD-1 ligand into an antigen presenting cell. 1 transmits an inhibitory signal to an immune cell in a manner similar to CTLA.4 Modulation of an inhibitory signal in an immune cell results in the modulation of the proliferation of, and / or secretion of cytokines by, an immune cell. , the expression "PD-1 activity" includes the ability of a PD-1 polypeptide to bind to its natural ligand (or ligands), the ability to modulate costimulatory or immune cell inhibitory signals and the ability to modulate the response immune

As used herein, the term "interaction", when referring to the physical contact (eg, union) of the molecules with each other. Generally, such interaction results in an activity (which produces a biological effect) of a or both of said molecules The activity may be a direct activity of one or both of the molecules (eg, transduction signal).

Alternatively, the binding of one or both molecules in the interaction to a ligand can be prevented and therefore remain inactive with respect to the ligand binding activity (for example, by binding to its ligand and triggering or inhibiting costimulation). The inhibition of such interaction results in the alteration of the activity of one or more molecules involved in the interaction. To enhance said interaction is to prolong or increase the probability of said physical contact and to prolong or increase the probability of said activity.

As used herein, the singular forms "a", "one / one" and "the" include plural referents unless the context clearly dictates otherwise.

It is understood that aspects and embodiments of the invention described herein include "which consist" and / or "which essentially consist of" aspects and embodiments.

Various aspects of the invention are described in more detail in the following subsections.

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I. Isolated nucleic acid molecules

One aspect of the invention relates to isolated nucleic acid molecules encoding polypeptides of the present invention (for example, those in Figures 4-5) or biologically active parts thereof, as well as nucleic acid fragments sufficient for use. as hybridization probes to identify nucleic acid molecules encoding these polypeptides and fragments for use as PCR primers for amplification or mutation of nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA generated using nucleotide analogs The nucleic acid molecule can be single or double stranded, but preferably it is double stranded DNA.

The term "isolated nucleic acid molecule" includes nucleic acid molecules that are separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. For example, with respect to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid molecule lacks sequences that naturally flank the nucleic acid (ie, sequences located at the 5 'and 3' ends of the nucleic acid molecule) in the genomic DNA of the organism from which the organism originates. nucleic acid. For example, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of any cellular material, or culture medium, when produced by recombinant techniques, or substantially devoid of chemical precursors or other chemicals. when it is chemically synthesized.

A nucleic acid molecule of the present invention (for example, those of Figures 4-5), or a part thereof, can be isolated using conventional molecular biology techniques and sequence information provided herein. For example, a nucleic acid molecule that includes all or a portion of sequences shown in Figures 4-5 can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed as a function of the sequences shown in Figures. Four. Five.

A nucleic acid molecule of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to conventional PCR amplification techniques. The nucleic acid molecule thus amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Additionally, oligonucleotides corresponding to nucleic acid sequences of the invention can be prepared by conventional synthesis techniques, for example, using an automatic DNA synthesizer.

In another embodiment, a nucleic acid molecule isolated from the invention comprises a nucleic acid molecule that is a complement to a nucleic acid molecule of the present invention (eg, those in Figures 4-5), or a part of the same. A nucleic acid molecule that is complementary to a nucleic acid molecule of the present invention (for example, that of Figures 4-5), or a part thereof, is one that is sufficiently complementary to the nucleotide sequence shown in Figures 4-5, such that it can hybridize with the respective nucleotide sequence shown in Figures 4-5, thereby forming a stable duplex.

An isolated nucleic acid molecule comprising a nucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, is disclosed herein. 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in Figures 27, or a part of any of these nucleotide sequences.

Additionally, the nucleic acid molecule of the invention may comprise only a part of a nucleic acid molecule of the present invention (for example, those of Figures 4-5), or a part thereof, for example, a fragment which can be used as a probe or primer or a fragment encoding a part of a polypeptide of the invention, for example, those in Figures 4-5. The probe / primer typically comprises substantially purified oligonucleotides. The oligonucleotide normally comprises a region of nucleotide sequence that hybridizes under stringent conditions with at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65 or 75 nucleotides consecutive of a nucleic acid molecule of the present invention (for example, those of Figures 4-5); of an antisense sequence of a nucleic acid molecule of the present invention (for example, those of Figures 4-5); or of a mutant of a nucleic acid molecule of the present invention (for example, those of Figures 4-5).

Probes based on a nucleic acid molecule of the present invention (for example, those of Figures 45) can be used to detect transcripts or genomic sequences encoding the same polypeptides or homologs. In an embodiment described herein, the probe further comprises a marker group attached thereto, for example, the marker group may be a radioisotope, a fluorescent compound, an enzyme or an enzymatic cofactor.

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A nucleic acid fragment encoding a "biologically active part of a polypeptide of the invention" can be prepared by isolating a part of the nucleotide sequence of a nucleic acid molecule of the present invention (for example, that of Figures 4-5 ) encoding a polypeptide having a biological activity of a polypeptide of the invention (for example, the ability to bind to its antigenic target), expressing the encoded part of the polypeptide of the invention (for example, by in vitro recombinant expression) and evaluating the activity of the encoded part of the polypeptide of the invention.

The invention further includes nucleic acid molecules that differ from the nucleotide sequence (or sequences) shown in Figures 4-5 due to the degeneracy of the genetic code, and therefore encode the same polypeptides as those encoded by the nucleotide sequence. respective shown in Figures 4-5. In another embodiment, a nucleic acid molecule isolated from the invention has a nucleotide sequence encoding a polypeptide of the present invention (eg, those in Figures 4-5).

Nucleic acid molecules corresponding to homologs of a nucleic acid molecule of the present invention (for example, those in Figures 4-5) can be isolated based on their homology with the nucleic acids disclosed herein using the cDNAs disclosed. herein, or a part thereof, as a hybridization probe according to conventional hybridization techniques under stringent hybridization conditions.

Accordingly, in another embodiment, a nucleic acid molecule isolated from the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions with the nucleic acid molecule comprising a nucleic acid molecule. of the present invention (for example, those in Figures 4-5).

As used herein, the expression "hybridized under stringent conditions" is intended to describe hybridization and washing conditions in which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that the sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to one another. Those skilled in the art know such stringent conditions and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. In Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press , Cold Spring Harbor, NY (1989), chapters 7, 9 and 11 may find additional stringent conditions A non-limiting example of stringent hybridization conditions includes 4x or 6x sodium chloride / sodium citrate (SSC) hybridization at about 65-70 ° C (or 4x SSC hybridization plus 50% formamide at about 42-50 ° C) followed by one or more washes in 1x SSC, at about 65-70 ° C. A further non-limiting example of stringent hybridization conditions includes hybridization in 6x SSC at 45 ° C, followed by one or more washes in 0.2x SSC, 0.1% SDS at 65 ° C. A non-limiting example of very stringent hybridization conditions includes hybridization in 1x SSC, at about 65-70 ° C (or hybridization in 1x SSC plus 50% formamide at about 42-50 ° C), followed by one or more washes in SSC 0.3x at about 65-70 ° C. A non-limiting example of hybridization conditions of reduced stringency includes hybridization in 4x or 6x SSC, at about 50-60 ° C (or alternatively hybridization in 6x SSC plus 50% formamide at about 40-45 ° C) followed by one or more washed in 2x, at approximately 50-60 ° C. The intermediate ranges of the values indicated above, for example, at 65-70 ° C or at 42-50 ° C are also intended to be included in the present invention. The SSPE (1x SSPE in 0.15M NaCl, 10mM NaH2PO4 and 1.25mM EDTA, pH 7.4) can be substituted with SSC (1x SSC is 0.15M NaCl and 15mM sodium citrate) in hybridization buffers and washing; Washing is done for 15 minutes after the end of hybridization. The hybridization temperature for hybrids that is expected to be less than 50 base pairs in length should be 5-10 ° C less than the melting temperature (Tf) of the hybrid, where Tf is determined according to the following equations. For hybrids smaller than 18 base pairs in length, Tf (° C) = 2 (No. of bases A + T) +4 (No. of bases G C). For hybrids between 18 and 49 base pairs in length, Tf (° C) = 81.5 + 16.6 (logio [Na +]) +0.41 (% G + C) - (600 / N), where N it is the number of bases in the hybrid; and [Na +] is the concentration of sodium ions in the hybridization buffer ([Na +] for 1x SSC = 0.165M). The person skilled in the art will also recognize that additional reagents can be added to the hybridization and / or wash buffers to decrease the nonspecific hybridization of nucleic acid molecules with membranes, for example, nitrocellulose or nylon membranes, including, but not limited to, blocking agents (for example, BSA or salmon or herring sperm transport DNA), detergents (for example, SDS), chelating agents (for example, EDTA), Ficoll, PVP and the like. When nylon membranes are used, in particular, an additional non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65 ° C, followed by one or more washes in 0.02M NaH2PO4, 1% SDS at 65 ° C, see, for example, Church and Gilbert (1984) Proc. Natl Acad. Sci. USA 81: 1991-1995 (or, alternatively, 0.2x SSC, 1% SDS).

The person skilled in the art will also appreciate that changes can be introduced by mutation in a nucleic acid molecule disclosed herein (for example, those in Figures 2-7), thus leading to changes in the amino acid sequence of the polypeptides encoded disclosed herein, without altering the functional capacity of the polypeptides. For example, nucleotide substitutions leading to amino acid substitutions in "non-essential" amino acid residues can be made in a nucleic acid molecule disclosed herein (for example, those in Figures 2-7). A "non-essential" amino acid residue is

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a moiety that can be altered from a nucleic acid molecule disclosed in the present invention (for example, those in Figures 2-7) without altering the biological activity, while for the biological activity a "essential" amino acid is required. For example, amino acid residues that are conserved among the polypeptides disclosed herein, for example, those that are required for the union of the polypeptides with their target antigen, are expected to be particularly poorly susceptible to alteration.

Accordingly, nucleic acid molecules encoding polypeptides (eg, those in Figures 2-7) that contain changes in amino acid residues that are not essential for activity are also disclosed herein. Such polypeptides differ in the amino acid sequence of those in Figures 2-7, but retain biological activity. In an embodiment disclosed herein, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to those in Figures 2-7.

An isolated nucleic acid molecule encoding an identical polypeptide to the polypeptides of Figures 2-7, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of those in Figures 2-7, of such such that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced into the nucleic acid molecules disclosed herein (eg, those in Figures 2-7) by conventional techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In one embodiment, conservative amino acid substitutions are made on one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues that have similar side chains have been identified in the art. These families include amino acids with basic side chains (for example, lysine, arginine, histidine), acid side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, asparagine, glutamine, serine, threonine, tyrosine, cistern), non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and side chains aromatics (for example, tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue predicted in a polypeptide disclosed herein (eg, those in Figures 2-7) can be replaced by another amino acid residue of the same side chain family. Alternatively, random mutations may be introduced along all or part of one or more nucleic acid molecules (eg, those in Figures 2-7), such as by saturation mutagenesis, and the resulting mutants can be screened with regarding its biological activity to identify mutants that retain activity. After mutagenesis of a nucleic acid molecule (for example, those in Figures 2-7), the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined.

As described herein, a mutant polypeptide can be tested for the ability to bind to and / or modulate the activity of natural PD-1 (eg, PD-1 ligands) or a partner of the PD-1 ligand ( for example, PD-1 and B7-1), modulate intra or intercellular signaling, modulate activation of T lymphocytes and / or modulate the immune response of an organism.

Also disclosed herein are isolated nucleic acid molecules encoding fusion proteins. Said nucleic acid molecules comprising at least a first nucleotide sequence encoding a disclosed polypeptide herein (eg, those in Figures 2-7) operatively linked to a second nucleotide sequence encoding a disclosed polypeptide in the This document (for example, those in Figures 2-7) can be prepared by conventional recombinant DNA techniques.

The expression characteristics of a nucleic acid molecule of the present invention (for example, those of Figures 4-5) in a cell line or microorganism can be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or in cloned microorganisms such that the inserted regulatory element is operatively linked with the nucleic acid molecules of the present invention (eg, those in Figures 4-5). For example, a heterologous regulatory element can be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with a nucleic acid molecule of the present invention (eg, those in Figures 4-5), using techniques , such as directed homologous recombination, which are well known to those skilled in the art, and are described, for example, in Chappel, US Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

II. Isolated polypeptide molecules

One aspect of the invention relates to isolated polypeptides of the present invention (including antibodies and antigen binding fragments thereof described herein, and those of Figures 4-5) and biologically active parts thereof. In one embodiment, the polypeptides of the present invention (eg, those in Figures 4-5), and biologically active parts thereof can be isolated from cell or tissue sources by an appropriate purification scheme using protein purification techniques.

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conventional. In another embodiment, the polypeptides of the present invention (for example, those in Figures 4-5) and biologically active portions thereof are produced by recombinant DNA techniques. Alternatively, the polypeptides of the present invention (for example, those of Figures 4-5), and biologically active portions thereof can be chemically synthesized using conventional peptide synthesis techniques.

An "isolated" or "purified" polypeptide or biologically active part thereof substantially lacks cellular material or other contaminating proteins from the cellular or tissue source from which the polypeptides of the present invention originate (eg, those in Figures 4- 5), or substantially lacks chemical precursors or other chemicals when chemically synthesized. The phrase "substantially lacks cellular material" includes preparations of one or more polypeptides of the present invention (for example, those in Figures 4-5), and biologically active portions thereof, in which the polypeptides are separated from Cellular components of the cells from which it is recombinantly produced or isolated. In one embodiment, the phrase "substantially lacks cellular material" includes preparations of one or more polypeptides of the present invention (eg, those in Figures 4-5), and biologically active portions thereof that have a smaller amount of approximately 30% (by dry weight) of proteins that are not of the present invention (also referred to herein as a "contaminating protein"), more preferably an amount less than about 20% of proteins that are not of the present invention, even more preferably an amount of protein less than about 10% that is not of the present invention, and most preferably an amount of protein less than about 5% that is not of the present invention. When the polypeptides of the present invention (for example, those of Figures 4-5) or a biologically active part thereof are produced recombinantly, it is also preferably that it lacks substantially culture medium, that is, the medium of culture represents less than about 20%, more preferably less than about 10% and most preferably less than about 5% of the volume of the protein preparation.

The expression "substantially lacks chemical precursors or other chemical products" includes preparations of one or more polypeptides of the present invention (eg, those in Figures 4-5), or a biologically active part thereof in which the The polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the phrase "substantially lacks chemical precursors or other chemicals" includes preparations of one or more polypeptides of the present invention (eg, those in Figures 4-5) or biologically active part thereof which have a less than about 30% (dry weight) amount of chemical precursors or proteins that are not of the present invention, more preferably a less than about 20% amount of chemical precursors or proteins not of the present invention, even more preferably a less than about 10% amount of chemical precursors or proteins that are not of the present invention, and most preferably a less than about 5% amount of chemical precursors or proteins that are not of the present invention.

As used herein, a "biologically active part" of one or more polypeptides of the present invention (eg, those in Figures 4-5), include polypeptides that participate in an interaction between a PD-L1 molecule and a non-PD-L1 molecule, for example, a natural ligand of PD-1 ligands, for example, PD-1 or B7-1, respectively. Biologically active portions of one or more polypeptides of the present invention (for example, those of Figures 4-5) include peptides comprising amino acid sequences sufficiently identifiable to or from the amino acid sequence of one or more polypeptides of the present invention. (for example, those of Figures 4-5), which include less amino acids compared to one or more respective full-length polypeptides of the present invention (for example, those of Figures 4-5), and exhibit at least one activity of the respective polypeptide (or polypeptides) of the present invention (eg, those in Figures 4-5). In one embodiment, biologically active portions comprise a domain or motif with the ability to specifically bind PD-1 or a PD-L1 ligand according to the antigen, respectively, with which it is raised or designed to bind. The biologically active parts of one or more polypeptides of the invention (eg, those in Figures 4-5) can be used as targets for the development of agents that modulate an activity mediated by PD-1 or PD-L1, for example, activation or suppression of immune cells.

In one embodiment, one or more polypeptides of the present invention (eg, those in Figures 4-5) has an amino acid sequence shown in Figures 4-5. It is also described herein that the polypeptide is substantially identical to one or more polypeptides shown in Figures 2-7 and retains the functional activity of the one or more respective polypeptides shown in Figures 2-7, but differs in sequence. of amino acids due to mutagenesis, as described in detail in subsection 1 above. Accordingly, a polypeptide comprising an amino acid sequence at least about 71%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% is also described herein. , 96% 97%, 98%, 99%, 99.5% or 99.9% or more identical to one or more polypeptides shown in Figures 2-7.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps may be introduced in one or both of a first and a second amino acid sequence or Nucleic acid for optimal alignment may not be considered sequences that are not identical for comparative purposes.) As described in the

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herein, the length of a reference sequence aligned for comparative purposes has a length of at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60% and even more preferably at least 70 %, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the length of the reference sequence. Then, amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid or nucleotide moiety as the corresponding position in the second sequence, then the molecules are identical in that position (as used herein, an "identity" of amino acid or nucleic acid is equivalent to a "homology" of amino acid or nucleic acid). The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of holes and the length of each hole, which needs to be introduced for the optimal alignment of the two sequences.

The disclosure also provides chimeric or fusion proteins. As used herein, a "chimeric protein" or "fusion protein" comprises one or more polypeptides of the present invention (eg, those in Figures 4-5) operatively linked to a polypeptide that is not of the present invention. A "polypeptide (polypeptides) of the present invention" refers to a polypeptide having an amino acid sequence corresponding to a polypeptide shown in Figures 4-5, while a "polypeptide that is not of the present invention" refers to a polypeptide that does not have an amino acid sequence corresponding to a polypeptide that is not substantially homologous to a polypeptide shown in Figures 4-5, for example, a polypeptide that is different from a polypeptide shown in Figures 4-5 and that proceeds from the same organism or from a different organism. Within the fusion protein, the expression "operably linked" is intended to indicate that the polypeptide (or polypeptides) of the present invention and that the polypeptide (or polypeptides) not described in the present invention are fused in phase with each other. The polypeptide or polypeptides that are not of the present invention can be fused with the N-terminus or C-terminus of the polypeptide (or polypeptides) of the present invention and corresponds to a fraction that alters the solubility, binding affinity, stability or valence of the polypeptide ( or polypeptides) of the present invention.

For example, in one embodiment, the fusion protein is a GST fusion protein with a polypeptide (or polypeptide) of the present invention. Such fusion proteins may facilitate purification of the recombinant polypeptides of the invention. In another embodiment, the fusion protein contains a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian host cells), the expression and / or secretion of the polypeptide (or polypeptides) of the present invention may be increased through the use of a heterologous signal sequence.

A chimeric or fusion polypeptide (or polypeptide) of the present disclosure (eg, those in Figures 2-7) can be produced by conventional recombinant DNA techniques. For example, DNA fragments encoding the different polypeptide sequences are linked to each other in phase according to conventional techniques, for example, using blunt or stepped end terms for ligation, digestion with restriction enzymes to provide appropriate ends, filling cohesive ends as appropriate, treatment with alkaline phosphatase to prevent undesirable binding, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automatic DNA synthesizers. Alternatively, PCR amplification of genetic fragments can be performed using anchor primers that give rise to complementary projections between two consecutive genetic fragments that can subsequently be hybridized and reamplified to generate a chimeric genetic sequence (see, for example, Current Protocols in Molecular Biology , Ausubel et al., Eds., John Wiley & Sons: 1992). In addition, many expression vectors are commercially available that already encode a fusion moiety (for example, a GST polypeptide).

The amino acid sequences of the polypeptide (or polypeptides) of the present invention (for example, those of Figures 4-5) identified herein will enable those skilled in the art to produce polypeptides corresponding to the polypeptide (or polypeptides) herein. invention (for example, those in Figures 4-5). Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding one or more polypeptides of the present invention (eg, those in Figures 4-5). Alternatively, said peptides can be synthesized by chemical methods. Methods for the expression of heterologous polypeptides in recombinant hosts, chemical synthesis of polypeptides and in vitro translation are well known in the art and are further described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, NY; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif .; Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501; Chaiken I. M. (1981) CrC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243: 187; Merrifield, B. (1986) Science 232: 342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57: 957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing).

III. Antibodies against PD-L1

The antibodies against PD-L1 described herein can be produced using any of the methods described herein or known in the art. Monoclonal antibodies (for example,

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Human antibodies) of the invention can be produced using various known techniques, such as the conventional somatic cell hybridization technique described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques can also be employed for the production of monoclonal antibodies, for example, vmca or oncogenic transformation of B lymphocytes, phage display technique using human antibody gene libraries.

One method of generating hybridomas that produce monoclonal antibodies of the invention is the murine system. The production of hybridomas in the mouse is well known in the art, including protocols and immunization techniques for the isolation and fusion of splenocytes.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide immunogen. The titration of the polypeptide antibody in the immunized subject can be monitored over time by conventional techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using an immobilized polypeptide. If desired, the antibody directed against the antigen can be isolated from the mammal (for example, from the blood) and further purified by well-known techniques, such as chromatography with protein A to obtain the IgG faction. At an appropriate time after immunization, for example, when the antibody titres are higher, the antibody producing cells can be obtained from the subject and used to prepare monoclonal antibodies by conventional techniques, such as the hybridoma technique originally described by Kohler. and Milstein (1975) Nature 256: 495-497 (see also Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol. Chem. 255: 4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. 76: 2927-31; and Yeh et al. (1982) Int. J. Cancer 29: 269-75), the human B lymphocyte hybridoma technique more Recent (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 ) or trioma techniques. The technology to produce monoclonal antibody hybridomas is well known (see in general Kenneth, RH in Monoclonal Antibodies: A New Dimension In Biological Analyzes, Plenum Publishing Corp., New York, New York (1980); Lerner, EA (1981) Yale J. Biol. Med. 54: 387-402; Gefter, ML et al. (1977) Somatic Cell Genet. 3: 231-36). In summary, an immortal cell line (usually a myeloma) is fused with lymphocytes (usually splenocytes) of a mammal immunized with an immunogen as described above, and the culture supernatant of the resulting hybridoma cells is screened to identify a producing hybridoma. of a monoclonal antibody that binds to the polypeptide antigen, preferably in a specific way.

Any of the many well-known protocols used to fuse immortalized lymphocytes and cell lines can be applied for the purpose of generating an anti-PD-L1 monoclonal antibody (see, for example, Galfre, G. et al. (1977) Nature 266: 55052 ; Gefter et al. (1977) cited above; Lerner (1981) cited above; Kenneth (1980) cited above). In addition, the usual expert will appreciate that there are many variations of these methods that would also be useful. Normally, the immortal cell line (for example, a myeloma cell line) comes from the same mammalian species as that of lymphocytes. For example, murine hybridomas can be prepared by fusing lymphocytes of an immunized mouse with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortalized cell lines are mouse myeloma cell lines that are sensitive to culture media containing hypoxanthine, aminopterin and thymidine ("HAT medium"). As a fusion partner, any of several myeloma cell lines may be used according with conventional techniques, for example, the myeloma lines P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag.14 These myeloma lines are available in the American Type Culture Collection ( ATCC), Rockville, Md. Normally, HAT-sensitive mouse myeloma cells are fused with mouse splenocytes using polyethylene glycol ("PEG"). The hybridoma cells resulting from the fusion are then selected using hAt medium, which destroys the non-fused and fused myeloma cells in a non-productive manner (the non-fused splenocytes die after several days because they do not transform). Hybridoma cells producing a monoclonal antibody of the invention are detected by scanning the hybridoma culture supernatants for antibodies that bind to a particular polypeptide, for example, using a conventional ELISA.

As an alternative to the preparation of hybridomas that secrete monoclonal antibodies, a specific monoclonal antibody can be identified and isolated for one of the polypeptides described above by exploring a recombinant combinatorial immunoglobulin library (eg, an antibody phage library) with the appropriate polypeptide to isolate thus the members of the immunoglobulin library that bind to the polypeptide. Commercially available kits to generate and explore phage display libraries (for example, Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; and Stratagene's SurfZAP ™ phage display kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly suitable for use in the generation and exploration of a phage display library, for example, Ladner et al. US Patent 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al International Publication No. WO 92/20791; Markland et al. International Publication No. WO 92/15679; Breitlina et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY) 9: 1369-1372; There et al. (1992) Hum. Antibody Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J. 12: 725-734; Hawkins et al. (1992) J. Mol. Biol. 226: 889-896;

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Clarkson et al. (1991) Nature 352: 624-628; Gram et al. (1992) Proc. Natl Acad. Sci. USA 89: 3576-3580; Garrard et al. (1991) Biotechnology (NY) 9: 1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137; Barbas et al. (1991) Proc. Natl Acad. Sci. USA 88: 7978-7982; and McCafferty et al. (1990) Nature 348: 552-554.

Additionally, recombinant PD-L1 antibodies, such as compound and humanized monoclonal antibodies, can be generated which can be prepared using conventional recombinant DNA techniques. Such compound and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, using methods described in Robinson et al. International Patent Publication WO 87/002671; Akira et al. European Patent Application 184,187; Taniguchi, M. European Patent Application 171,496; Morrison et al. European patent application 173,494; Neuberger et al. PCt application WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European patent application 125,023; Better et al. (1988) Science 240: 1041-1043; Liu et al. (1987) Proc. Natl Acad. Sci. USA 84: 3439-3443; Liu et al. (1987) J. Immunol. 139: 3521-3526; Sun et al. (1987) Proc. Natl Acad. Sci. 84: 214-218; Nishimura et al. (1987) Cancer Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559); Morrison, S. L. (1985) Science 229: 1202-1207; Oi et al. (1986) Biotechniques 4: 214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol. 141: 4053-4060.

In addition, humanized antibodies can be prepared according to conventional protocols such as those disclosed in US Patent 5,565,332. In another embodiment, antibody chains or specific binding pair members may be produced by recombination between vectors comprising nucleic acid encoding a fusion of a polypeptide chain of a specific binding pair member and a component of a replicable generic presentation packaging and vectors containing nucleic acid molecules encoding a second polypeptide chain of a single binding pair member using techniques known in the art, for example, as described in US Patent 5,565,332, 5,871,907 or 5,733 .743. The use of intracellular antibodies that inhibit the function of proteins in a cell is also known in the art (see, for example, Carlson, JR (1988) Mol. Cell. Biol. 8: 2638-2646; Biocca, S. et al. (1990) EMBO J. 9: 101-108; Werge, TM et al. (1990) FEBS Lett. 274: 193-198; Carlson, JR (1993) Proc. Natl. Acad. Sci. United States 90: 7427-7428; Marasco, WA et al. (1993) Proc. Natl. Acad. Sci. United States 90: 7889-7893; Biocca, S. et al. (1994) Biotechnology (NY) 12: 396-399; Chen , SY. Et al. (1994) Hum. Gene Ther. 5: 595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. United States 91: 5075-5079; Chen, SY. Et al. (1994) Proc. Natl. Acad. Sci. United States 91: 5932-5936; Beerli, RR et al. (1994) J. Biol. Chem. 269: 23931-23936; Beerli, RR et al. (1994 ) Biochem. Biophys. Res. Commun. 204: 666-672; Mhashilkar, AM et al. (1995) EMBO J. 14: 15421551; Richardson, JH et al. (1995) Proc. Natl. Acad. Sci. United States 92: 3137-3141; PCT Publication No. WO 94 / 02610 by Marasco et al .; and PCT Publication No. WO 95/03832 by Duan et al.).

In another embodiment, human monoclonal antibodies directed against PD-L1 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system instead of the mouse system. In one embodiment, transgenic mice, referred to herein as "HuMAb mice" that contain a miniloci of human immunoglobulin gene that encodes unregulated human light and heavy (| jyy) light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous | jyk chain loci (Lonberg, N. et al. (1994) Nature 368 (6474): 856 859) .Therefore, mice exhibit reduced mouse IgM expression ok, and in response to immunization, transgenes Human light and heavy chain introduced are subjected to class modification and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), cited above; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49 101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93, and Harding, F. and Lonberg, N. (1995) Ann. N. And Acad. Sci 764: 536 546) .The preparation of HuMAb mice is described e in Taylor, L. et al. (1992) Nucleic Acids Research 20: 6287 6295; Chen, J. et al. (1993) International Immunology 5: 647 656; Tuaillon et al. (1993) Proc. Natl Acad. Sci United States 90: 3720 3724; Choi et al. (1993) Nature Genetics 4: 117 123; Chen, J. et al. (1993) EMBO J. 12: 821 830; Tuaillon et al. (1994) J. Immunol. 152: 2912 2920; Lonberg et al., (1994) Nature 368 (6474): 856 859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49 101; Taylor, L. et al. (1994) International Immunology 6: 579 591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65 93; Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764: 536 546; Fishwild, D. et al. (1996) Nature Biotechnology 14: 845 851. See also, US Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all of Lonberg and Kay, and international GenPharm; U.S. Patent No. 5,545,807 to Surani et al .; International Publication No. WO 98/24884, published June 11, 1998; WO 94/25585, published November 10, 1994; WO 93/1227, published June 24, 1993; WO 92/22645, published December 23, 1992; WO 92/03918, published March 19, 1992.

In another embodiment, an antibody for use in the invention is a bispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. The antigenic union can be simultaneous or sequential. Triomas and hybrids are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybridoma hybridoma or a trioma are disclosed in US Patent No. 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314: 628, and Perez et al. (1985) Nature 316: 354) and technology

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hybridoma (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. United States, 83: 1453, and Staerz and Bevan (1986) Immunol. Today 7: 241). Bispedic antibodies are also described in U.S. Patent No. 5,959,084. In United States patent 5,798,229, bispedic antibody fragments are described. They can also. Generating bispedic agents preparing heterohybridomas by fusing hybridomas or other cells preparing different antibodies, followed by identification of clones that produce and co-assemble both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or parts thereof such as Fab and Fv sequences. The antibody component can bind to PD-L1.

Another aspect of the invention relates to anti-PD-L1 polypeptide antibodies that can be obtained by a process comprising immunizing an animal with an immunogenic PD-L1 polypeptide, respectively, or with an immunogenic part; and then isolate from the animal antibodies that specifically bind to the polypeptide.

In yet another aspect of the invention, partial or known antibody sequences can be used to generate and / or express new antibodies. Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain complementary complementarity determining (CDR) regions. For this reason, the amino acid sequences in the CDRs are more diverse among individual antibodies than sequences outside the CDRs. Since CDR sequences are responsible for the majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of naturally occurring specific antibodies by constructing expression vectors that include CDR sequences of the naturally occurring specific sequence grafted antibody. framework conserved from a different antibody with different properties (see, for example, Riechmann, L. et al., 1998, Nature 332: 323 327; Jones, P. et al., 1986, Nature 321: 522 525; and Queen, C. et al., 1989, Proc. Natl. Acad. See. United States 86: 10029 10033). Such conserved framework sequences can be obtained from public DNA databases that include germline or non-germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they do not include fully assembled variable genes, which are formed by V (D) J binding during maturation of B lymphocytes. Germline gene sequences will also differ from those Sequences of a high affinity secondary repertoire antibody in individuals homogeneously across the variable region. For example, somatic mutations are relatively rare in the amino-terminal part of the conserved framework region. For example, somatic mutations are relatively rare in the amino-terminal part of the conserved framework region 1 and in the carboxyl terminal part of the conserved framework region 4. In addition, many somatic mutations do not significantly modify the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody to recreate an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/045962 published September 16, 1999) . The partial light and heavy chain sequence that encompasses the CDR regions is normally sufficient for this purpose. The partial sequence is used to determine which germline and / or non-germline variable and union of genetic segments contributes to the recombinant antibody variable genes. The germline and / or non-germline sequence is then used to fill in missing parts of the variable regions. The heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by linkage or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short, overlapping oligonucleotides, and combined by PCR amplification to create a completely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. The process can also be used to explore sequence libraries encoding particular immunoglobulins in a species (eg, human) to design sequences encoding related immunoglobulins of known antibody sequences in other species (e.g., mouse) (see, for example , the examples section below).

The nucleotide sequences of heavy and light chain transcripts of a hybridoma are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capabilities as natural sequences. Synthetic heavy chain and kappa sequences can be differentiated from natural sequences in three ways: repeated nucleotide base chains are interrupted to facilitate oligonucleotide synthesis and PCR amplification; Optimal translation start sites are incorporated according to Kozak standards (Kozak, 1991, J. Biol. Chem. 266L19867019870); and HindIII sites are designed genetically upstream of the translation start sites.

For both the light and heavy chain variable regions, the sequences of the optimized coding chain, and corresponding non-coding, are degraded by approximately 30-50 nucleotides to the central point of the non-coding oligonucleotide. Therefore, for each chain, oligonucleotides can be assembled into overlapping double stranded assemblies that span 150-400 nucleotide segments. The assemblies are then used as molds to produce 150-400 nucleotide PCR amplification products. Normally, a single variable region oligonucleotide set will degrade into two sets that are amplified separately to generate two overlapping PCR products. These overlap products are then combined by PCR amplification to form the entire variable region. It may also be desirable to include an overlapping fragment of the light heavy chain constant region or PCR amplification to generate fragments that can

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Easily clone into expression vector constructs.

The reconstructed light and heavy chain variable regions are then combined with cloned promoter sequences, Kder sequences, translation start sequences, leader sequences, constant region, 3 'untranslated, polyadenylation, and transcription termination to form vector constructs expression. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected or transfected individually into host cells that are then fused to form a host cell that expresses both chains.

Plasmids are known for this use in the art and include the plasmids provided in the examples section below. The chimeric and fully human antibodies disclosed herein also include IgG2, IgG3, IgE, IgA, IgM and IgD antibodies. Similar plasmids can be constructed for the expression of other heavy chain isotypes, or for the expression of antibodies comprising lambda light chains.

Thus, in another aspect of the invention, the structural characteristics of known, non-human or human antibodies (for example, a mouse anti-human PD-LI antibody, such as antibody 29E.2A3) are used to create antibodies. Structurally related human anti-PD-LI that retain at least one functional property of the antibodies of the invention, such as binding with PD-L1. Another functional property includes inhibition of binding of 29E.2A3 with PD-L1 in a competition ELISA. In some embodiments, structurally related human anti-PD-LI antibodies have a lower binding affinity with the antigen compared to antibody 29E.2A3 as measured by the IC50 value as described in example 2 (for example, The affinity of the murine reference antibody is not greater than any of 3.0, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1 , 2 or 1.1 times of the structurally related antibody). In some embodiments, structurally related human anti-PD-L1 antibodies have a higher affinity for the antigen compared to antibody 29E.2A3 measured by the IC50 value as described in example 2 (such that the affinity of the antibody Structurally related is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 times of the reference antibody ). In addition, one or more CDR or variable regions of the present invention (eg, Figures 4-5) can be recombinantly combined with known human conserved framework regions and the CDRs create recombinantly modified human anti-PD-L1 antibodies, in addition to the invention.

Since it is well known in the art that the light and heavy chain CDR3 antibody domains play a particularly important role in the binding / affinity specificity of an antibody to an antigen, the recombinant antibodies of the invention prepared as set forth above comprise preferably the light and heavy chain CDR3 of the variable regions of the present invention (eg, Figures 4-5). Antibodies may additionally comprise CDR2 from variable regions of the present invention (eg, Figures 4-5). The antibodies may additionally comprise the CDR1 of the variable regions of the present invention (eg, Figures 4-5). The antibodies may additionally comprise any of the combinations of the CDRs.

The CDR1, 2 and / or 3 regions of the genetically modified antibodies described above may comprise the exact amino acid sequence (or sequences) such as those of the variable regions of the present invention (eg, Figures 4-5) disclosed herein. document.

In addition to simply binding PD-L1, genetically encoded antibodies such as those described above can be selected with respect to their retention of other functional properties of antibodies of the invention, such as:

(1) binding to human PD-L1;

(2) inhibition of the binding of 29E.2A3 with PD-L1;

(3) binding with human PD-L1 and inhibiting the ability in which bound PD-L1 binds to PD-L1 ligands (By

example, PD-1 and / or B7-1);

The heavy and light chain variable region amino acid sequences for the EH12.2H7, 29E.2A3 and 24F.10C12 antibodies are shown below.

EH12.2H7 heavy chain variable region

QVQLQQSGAELAKPGASVQMSCKASGYSFTSSWIHWVKQRPGQGLEWIGYIYPSTGFT EYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARWRDSSGYHAMDYWGQG TSVTVSS (SEQ 76):

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LH chain variable region of EH12.2H7

DIVLTQSPASLTVSLGQRATISCRASQSVSTSGYSYMHWYQQKPGQPPKLLIKFGSNLES GIPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPYTFGGGTKLEIK (SEQ ID NO: 77)

Variable region string of 29E.2A3

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKFGQGLEWIGYVNPFNDG TKYNEMFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARQAWGYFWGQGTLVTVS A (SE)

Region variable chain of 29E.2A3

DIVLTQSPASLAVSLGQRATISCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDS GVPARFSGSGSGTDFSLTIHPVEEDDIAMYFCQQSRRVPYTFGGGTKLEIK (SEQ ID NO: 79)

Variable heavy chain region of 24F.10C12

QVQLQQSAAELARPGASVKMSCKASGYTFTGYTMHWVKQRPGQGLEWIGY1NPRSGY I'EYNQKFKDK'I'I LI ADKSSS'I AYMQLSSL'I SEDSAVYYCARPWFAYWGQG 1'LV'I VD: 80

Variable region of the chain of 24F.10C12

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELK (SEQ ID NO: 81)

The activity of the antibodies in inhibiting the binding of PD-L1 with its ligand (or ligands) can be determined by testing the ability of the antibody to block the binding between PD-L1 and its ligand. A competition ELISA assay can be used in the presence of a labeled ligand and the antibody. For example, to determine if an anti-PD-LI antibody could block the interaction between PD-1 and PD-L1, a competitive binding experiment is performed. Cells expressing PD-L1 are pre-incubated with the anti-PD-LI antibody followed by the addition of the biotinylated PD-1-Ig fusion protein. If the anti-PD-LI antibody blocks the binding of PD-1-Ig in a dose-dependent manner and with high avidity, the anti-PD-LI antibody is considered to be effective in inhibiting interaction between pD -1 and PD-L1.

IV. Recombinant expression vectors and host cells

Another aspect of the invention relates to vectors, preferably, to expression vectors, which contain one, two or more nucleic acid molecules encoding one or more polypeptides of the present invention (eg, Figures 4-5) (or a part of them). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been attached. One type of vector is a "plasmid," which refers to a DNA loop. circular double stranded in which additional segments of DNA can be ligated Another type of vector is a viric vector, in which additional segments of DNA can be ligated into the virome genome Certain vectors are capable of replicating autonomously in a host cell in which are introduced (for example, bacterial vectors that have an origin of bacterial replication and episomic mammalian vectors) Other vectors (for example, non-episomal mammalian vectors) are integrated into the genome of a host cell after introduction into the host cell, and therefore they replicate together with the host genome.In addition, certain vectors are capable of directing the expression of genes with which they are linked s operatively. In this document, said vectors are called "expression vectors". In general, expression vectors useful in recombinant RNA techniques are often in the form of plasmids. In the present specification, "plasmid" and vector "can be used interchangeably since the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of this type of expression vectors, such as viric vectors (for example,

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retrovirus with defective replication, adenoviruses and adeno-associated viruses), which perform equivalent functions.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for the expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected according to the host cells that are used for expression, which is operatively linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to one or more regulatory sequences in a manner that allows the expression of the nucleotide sequence (for example, in a transcription system / translation in vitro or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185: 3-7. Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many types of host cells and those that direct the expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein and the like. Expression vectors of the invention can be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors of the invention can be designed for the expression of polypeptides of the present invention (eg, Figures 4-5) in prokaryotic or eukaryotic cells. For example, polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further analyzed in Goeddel (1990) cited above. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

The expression of polypeptides in prokaryotes is most frequently performed in E. coli with vectors containing constitutive or inducible promoters that direct the expression of fusion or non-fusion proteins. The fusion vectors add various amino acids to a polypeptide encoded therein, usually at the amino terminus of the recombinant polypeptide. Such fusion vectors normally perform three purposes: 1) increase the expression of the recombinant polypeptide; 2) increase the solubility of recombinant polypeptide; and 3) assist in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in the fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion fraction and the recombinant polypeptide to allow separation of the recombinant polypeptide from the fusion fraction after purification of the protein from fusion. Such enzymes, and their related recognition sequences, include factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, NJ) that fuse glutathione S-transferase (GST), maltose binding protein E or protein A, respectively, to the recombinant target polypeptide.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69: 301-315) and pET 1 Id (Studier et al. (1990) Methods Enzymol. 185: 60-89). The expression of the pTrc vector target gene is based on the transcription of the host RNA polymerase from a hybrid trp-lac fusion promoter. The expression of the pET 11 vector target gene is based on the transcription of a T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gn1). This viral polymerase is provided by BL21 (DE3) or HMS174 (DE3) host cells of a resident profago that hosts a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

A strategy to maximize the expression of recombinant polypeptides in E. coli is to express the polypeptide in host bacteria with defective ability to proteolytically cleave the recombinant polypeptide (Gottesman, S. (1990) Methods Enzymol. 185: 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons of each amino acid are used preferentially in E. coli (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Said alteration of nucleic acid sequence acids of the invention can be carried out by conventional DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector. Examples of S. cerevisiae yeast expression vectors include pYepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) and picZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, the polypeptides of the present invention (eg, Figures 4-5) can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression

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Polypeptides in cultured insect cells (eg, Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170: 31-39).

In yet another embodiment, a nucleic acid of the present invention (eg, Figures 4-5) is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the functions of expression vector control are often provided by viral regulatory elements. For example, commonly used promoters come from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells see Chapters 16 and 18 of Sambrook, J. et al., Molecular Cloning : A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the expression vector of a recombinant mammal can direct the expression of the nucleic acid preferably in a particular type of cell (for example, tissue specific regulatory elements are used to express the nucleic acid). Specific tissue regulatory elements are known in the art. Non-limiting examples of suitable tissue specific promoters include the albumine promoter (liver specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), particular promoters of T-lymphocyte receptors (Winoto and Baltimore (1989) EMbO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen and Baltimore (1983) Cell 33: 741-748), specific neuron promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. United States 86: 5473-5477), promoters specific to pancreas (Edlund et al. (1985) Science 230: 912-916), and specific breast gland promoters (e.g., whey promoter; U.S. Patent No. 4,873,316 and European application publication No. 264,166). Also included are promoters regulated by evolution, for example, by murine hox promoters (Kessel and Gruss (1990) Science 249: 374-379) and the alpha fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546) .

Another aspect of the invention relates to host cells in which a nucleic acid molecule of the present invention (eg, Figures 4-5) is introduced into a recombinant expression vector or a nucleic acid molecule containing sequences that allow recombine homologously at a specific site of the genome of the host cell. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that said expressions refer not only to the cell of the particular subject but also to the offspring or possible offspring of said cell. Because certain modifications may Occurring in successive generations due to mutation or environmental influences, such offspring may not be, in fact, identical to the parental cell, but still be included within the scope of the expression as used herein.

A host figure can be any prokaryotic or eukaryotic cell. For example, a polypeptide of the present invention (eg, Figures 4-5) can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) ) or COS). Those skilled in the art know other host cells that are suitable.

Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various techniques recognized in the art for introducing exogenous nucleic acid (eg, DNA) into a host cell, including co-precipitation with calcium phosphate. or calcium chloride, transfection mediated with DEAE-dextran, lipofeccion or electroporation. In Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals can find suitable methods for transforming or transfecting host cells.

For the stable transfection of mammalian cells, it is known that, depending on the expression vector and the transfection technique used, only a small fraction of cells can integrate the exogenous DNA into their genome. To identify and select these members, generally a gene encoding a selection marker (for example, antibiotic resistance) is introduced into the host cells along with the gene of interest. Preferred selection markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. The nucleic acid encoding a selection marker can be introduced into a host cell in the same vector as that encoding a PD-L1 polypeptide or can be introduced into a different vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (for example, cells that have the selection marker gene incorporated will survive, while the others will die).

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express), a polypeptide of the present invention (eg, Figures 4-5). Accordingly, the invention further provides methods for producing a polypeptide of the present invention (eg, Figures 4-5) using the host cells of the present invention. In one embodiment, the method

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it comprises culturing the host cell of the invention (into which a recombinant expression vector has been introduced that encodes a polypeptide of the present invention (eg, Figures 4-5)) in a suitable medium such that a polypeptide of the present invention (eg, figures 4-5). In another embodiment, the method further comprises isolating a polypeptide of the present invention (eg, Figures 4-5) from the medium or from the host cell.

The host cells of the invention can also be used to produce non-human transgenic animals, as described below.

V. Production of transgenic and transchromosomal non-human animals that generate human PD-L1 antibodies, compounds

In a further aspect, the invention provides transgenic and transchromosomal non-human animals, such as transgenic or transchromosomal mice, that can express human monoclonal antibodies that specifically bind PD-L1. In a particular embodiment with the invention it provides a transgenic or transchromosomal mouse that has a genome comprising a human heavy chain transgene, such that the mouse produces human anti-PD-LI antibodies when immunized with PD-L1 and / or antigen. or cells expressing PD-L1. The human heavy chain transgene can be integrated into the mouse chromosomal DNA as in the case of transgenic mice, for example, HuMAb, according to methods well known in the art. Alternatively, the human heavy chain transgene can be maintained extrachromosomically, as is the case with transchromosomal mice (eg, KM) as described in WO 02/43478. Such transgenic and transchromosomal mice can produce multiple isotypes of human monoclonal antibodies against PD-L1 (eg, IgG, IgA and / or IgE) by recombination V-D-J and isotype switching. The isotype change can occur through, for example, classical or non-classical isotype change.

The design of a transgenic or transchromosomal non-human animal that responds to exogenous antigenic stimulation with a repertoire of heterologous antibodies, requires that the heterologous immunoglobulin transgenes contained in the transgenic animal function properly along the path of B lymphocyte development. This it includes, for example, the isotype change of the heterologous heavy chain transgene. Therefore, transgenes are constructed to produce the change of isotypes and one or more of the following antibodies: (1) high level and specific expression of cell type, (2) functional genetic rearrangement, (3) activation of and response to exclusion allelic, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypermutation and (7) domination of the transgene antibody locus during the immune response.

It is not necessary that all the above criteria are met. For example, in those embodiments in which the endogenous immunoglobulin loci of the transgenic animal are functionally altered, the transgene does not require activating allelic exclusion. In addition, in those embodiments in which the transgene comprises a functionally rearranged heavy and / or light chain immunoglobulin gene, the second functional genetic rearrangement criterion is unnecessary, at least for that transgene that is already rearranged. For the background of molecular immunology, see, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y.

In certain embodiments, the transgenic or transchromosomal non-human animals used to generate the human monoclonal antibodies of the invention contain rearranged, non-rearranged heterologous light and heavy immunoglobulin transgenes or a combination of rearranged and unordered in the germ line of the transgenic animal. . Each of the heavy chain transgenes comprises at least one CH gene. In addition, the heavy chain transgene may contain functional isotype change sequences, which can support the isotype change of a heterologous transgene encoding multiple CH genes in the B lymphocytes of the transgenic animal. Said change sequences may be those that occur naturally in the germline immunoglobulin locus of the species that serves as a source for the transgenic CH genes, or such change sequences that may come from those that occur in the species that will receive the transgenic construction (the transgenic animal). For example, a human transgenic construction that is used to produce a transgenic mouse can produce a higher frequency of isotype change events if it incorporates similar change sequences to those that occur naturally in the mouse heavy chain locus. , since the mouse change sequences are supposedly optimized to act with the mouse exchange recombinase enzyme systems, while the human change sequences are not. The change sequences can be isolated and cloned by conventional donation methods or can be synthesized again from overlapping synthetic oligonucleotides designed based on published sequence information related to the sequences of the immunoglobulin change region (Mills et al., Nucl. Acids Res. 15: 7305 7316 (1991); Sideras et al., Intl. Immunol. 1: 631 642 (1989)). For each of the above transgenic animals, functionally rearranged heterologous light chain and heavy chain immunoglobulin transgenes have been discovered in a significant fraction of the B lymphocytes in the transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic animals of the invention include a heavy chain transgene comprising DNA encoding at least one variable genetic segment, a diversity genetic segment, a genetic binding segment and at least one constant region genetic segment. The transgene of

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immunoglobulin light chain comprises DNA that encodes at least one variable genetic segment, one genetic binding segment and at least one constant region genetic segment. The genetic segments encoding the light and heavy chain genetic segments are heterologous to the transgenic non-human animal in which they originate from, or correspond to, DNA encoding the immunoglobulin heavy and light chain genetic segments of a species that does not consist in the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual genetic segments are not rearranged, that is, not rearranged to encode a light or heavy chain of functional immunoglobulin. Said non-rearranged transgenes support recombination of the genetic segments V, D and J (functional rearrangement) and preferably support the incorporation of all or part of a genetic segment of region D in the resulting rearranged immunoglobulin heavy chain in the non-human animal transgenic when exposed to the PD-1, PD-L1 or PD-L2 antigen.

In an alternative embodiment, the transgenes comprise a non-rearranged "mini-locus." Such transgenes normally comprise a substantial part of the segments C, D and J, as well as a subset of the genetic segments V. In such transgenic constructions, the various regulatory sequences, for example, promoters, enhancers, class change regions, splice donor sequences and splice acceptors for RNA processing, recombination signals and the like, comprise corresponding sequences derived from heterologous DNA. Regulators may be incorporated into the transgene thereof or a related species of non-human animal used in the invention.For example, the immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, synthetic regulatory sequences can be incorporated into the transgene, and n that said synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in mammalian genomes. Synthetic regulatory sequences were designed according to consensus standards such as, for example, those that specify the permissible sequences of a splice acceptor site or a promoter / enhancer motif. For example, a minilocus comprises a part of the genomic immunoglobulin locus that has at least one internal deletion (i.e. not at one end of the part) of a part of nonessential DNA (e.g., intermediate sequence, intron or part of the same) in comparison with the Ig locus of the germline of natural origin.

The transgenic and transchromosomal mice employed in the present invention can exhibit immunoglobulin production with a significant repertoire, and ideally substantially similar to that of a natural mouse. Therefore, for example, in embodiments in which endogenous Ig genes have been inactivated, total immunoglobulin levels may vary from about 0.1 to 10 mg / ml of serum, or from about 0.5 to 5 mg / ml. , or at least about 1.0 mg / ml. When a transgene capable of eliciting a change to IgM from IgM has been introduced into the transgenic mouse, the ratio of adult mouse from serum IgG to IgM may be approximately 10: 1. The proportion of IgG with IgM will be much lower in the immature mouse. In general, more than about 10%, preferably 40 to 80% of spleen and lymph node lymphocytes can exclusively express the IgG protein.

The repertoire will be ideally approximated to that shown in a natural mouse, usually at least about 10% as high, or 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), for example, preferably from 104 to 106 or more, will be produced, depending mainly on the number of different regions V, J and D introduced into the mouse genome. These immunoglobulins normally recognize about half or more of highly antigenic proteins, for example, Staphylococcus protein A. Normally the immunoglobulins will exhibit an affinity (Kd) for preselected antigens of less than 10-7 M, such as less than 10-8 M, 10-9 M or 10-10 M or even lower.

In some embodiments, it may be preferable to generate mice with predetermined repertoires to limit the selection of V genes represented in antibody response against a predetermined antigenic type. A borrowed chain transgene having a predetermined repertoire may comprise, for example, human Vh genes that are preferably used in antibody responses against the predetermined type of antigen in humans. Alternatively, some Vh genes can be excluded from a repertoire defined for various reasons (for example, having a low probability of encoding high affinity V regions for the predetermined antigen; having a low propensity to experience somatic mutations and an affinity sharpening; or are immunogenic in certain human beings). Therefore, prior to the rearrangement of a transgene containing various heavy or light chain gene segments, said genetic segments can be easily identified, for example, by DNA hybridization or sequencing, as of species of organisms other than those of the transgenic animal. .

Transgenic and transchromosomal mice as described above can be immunized, for example, with a purified or enriched preparation of PD-L1 and / or cells expressing PD-L1. Alternatively, transgenic mice can be immunized with DNA encoding human PD-L1. The mice will then produce B lymphocytes that will undergo class change by intratransgenic exchange (cis-switching) recombination and express immunoglobulins reactive with PD-L1. Immunoglobulins may be human antibodies (also called "human sequence antibodies"), in which heavy and light chain polypeptides are encoded by human transgenic sequences, which may include sequences derived from somatic mutation and

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recombinant junctions of region V, as well as encoded sequences of the germ line; These human antibodies can be referenced by being substantially identical to a polypeptide sequence encoded by a Vl or Vh gene segment and a human Jl or Dh and Jh segment, even though other non-germline sequences may be present as a result of somatic mutation and binding. of differentiated recombination VJ and VDJ. The variable regions of each antibody chain are normally at least 80 percent encoded by genetic segments V, J, and, in the case of heavy chains, D, of human germline; frequently at least 85 percent of the variable regions are encoded by human germline sequences present in the transgene; frequently 90 or 95 percent or more of the variable region sequences are encoded by human germ line sequences in the transgene. However, since non-germline sequences are introduced by somatic mutation and VJ and VDJ binding, human sequence antibodies will often have some variable region sequences (and less frequently constant region sequences) that are not encoded by V gene segments, D or J as found in the human transgene or transgenes in the germ line of mice. Normally, said non-germline sequences (or individual nucleotide positions) will be grouped in or near the CDRs, or in regions where somatic mutations are known to be grouped.

Human antibodies that bind to the predetermined antigen can result in the change of isotype, such that human antibodies are produced that comprise a human chain and sequence (such as y1, Y2a, y2B or y3) and a light chain of human sequence (such as kappa). Such human isotype change antibodies often contain one or more somatic mutations normally in the variable region and often at or within about 10 CDR residues as a result of affinity maturation and selection of B lymphocytes for antigen, particularly post exposure. secondary (or later) antigenic. These highly related human antibodies may have binding affinities (Kd) of less than 10-7 M, such as less than 10-8 M, 10-9 M, 10-10 M, 10-10 M or even less.

Another aspect of the invention includes B lymphocytes from transgenic or transchromosomal mice as described herein. B lymphocytes can be used to generate hybridomas that express human monoclonal antibodies that bind with high affinity (eg, less than 10-7 M) to human PD-L1.

The development of high-affinity human monoclonal antibodies against PD-L1 can be facilitated by a method to expand the repertoire of human variable region gene sequences in a transgenic mouse having a genome comprising an integrated human immunoglobulin transgene, said method comprising introducing into the genome a genetic transgene V comprising genetic segments of region V that are not present in said integrated human immunoglobulin transgene. Often, the region V transgene is an artificial yeast chromosome that comprises a part of a human Vh or Vl (Vk) gene segment as can occur naturally in a human genome or can be cut and spliced together separately by recombinant methods , which may include disordered or omitted V gene segments. Often at least five or more functional V gene segments are contained in the YAC. In this variation, it is possible to make a transgenic mouse produced by the method of repertoire expansion V, in which the mouse expresses an immunoglobulin chain comprising a variable region sequence encoded by a genetic segment of region V present in the transgene of region V and a region C encoded in the human Ig transgene. Through the method of expanding V repertoires, transgenic mice can be generated that have at least 5 different V genes; as well as mice that contain at least about 24 V genes or more. Some V gene segments may be nonfunctional (eg, pseudogenes and the like); those segments may be retained or selectively deleted by recombinant methods available to the person skilled in the art if desired.

Once the mouse germ line has been modified by genetic engineering to contain a functional YAC that has an expanded V segment repertoire, substantially not present in the human Ig transgene containing the genetic segments J and C, the trait can propagate and reproduce in other genetic funds, including funds in which the YAC that has an expanded segment V repertoire is reproduced in a mouse germ line that has a different human Ig transgene. Multiple functional YACs that have an expanded V segment repertoire can be reproduced in a germ line to function with a human Ig transgene (or multiple human Ig transgenes). Although they are referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences necessary for autonomous replication in yeasts; such sequences can optionally be removed by genetic modification (for example, digestion with restriction enzymes and impulse field gel electrophoresis or other suitable method) after replication in yeasts that is no longer necessary (i.e., before introduction into a mouse ES cell or mouse procigote). Methods of propagation of the immunoglobulin expression trait of the human sequence include the reproduction of a transgenic mouse that has one or more human Ig transgenes and optionally also has a functional YAC that has an expanded V segment repertoire. Both Vh and Vl gene segments may be present in the YAC. The transgenic mouse can reproduce on any background desired by the specialist, including funds carrying other human transgenes, including human Ig transgenes and / or transgenes encoding other human lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse that has a YAC transgene with an expanded V region repertoire. Although the above

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describes a preferred embodiment of the transgenic animal of the invention, other embodiments that have been classified into four categories are contemplated:

(1) transgenic animals that contain a rearranged and heavy light immunoglobulin transgene not

rearranged

(2) transgenic animals containing a non-rearranged and heavy non-rearranged immunoglobulin transgene

rearranged

(3) transgenic animal that contains a transgene of non-rearranged and heavy rearranged immunoglobulin;

Y

(4) transgenic animals containing rearranged and heavy light immunoglobulin transgenes

rearranged

SAW. Antibody / Immunotoxin Conjugates

In another aspect, the present invention relates to human PD-L1 antibodies conjugated to a therapeutic fraction, such as a cytotoxin, a drug (eg, an immunosuppressant) or a radioisotope. When conjugated with a cytotoxin, these antibody conjugates are called "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is harmful to (for example, destroys) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emethine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mitramycin, actinomycin D-glucoside D-streptocorticide, glutathrotone, D-actinhydrocides D-glucoside D-actinhydrocides D-glucoside D-glucoside D-actinhydrocides , procaine, tetracaine, lidocaine, propanolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (for example, methotrexate, 6-mercaptopurine, 6-thioguaine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (for example, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (for example, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g. (formerly actinomycin), bleomycin, mitramycin, and antramycin (AMC)), and anti-mitotic agents (for example, vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, for example, radioactive iodine, to generate cytotoxic radiopharmaceuticals for the treatment of a related disorder, such as cancer.

Conjugated human PD-L1 antibodies can be used from the diagnostic or prognostic point of view to monitor levels of polypeptides in tissues as part of a clinical trial procedure, for example, to determine the efficacy of a particular treatment regimen. Detection can be facilitated by coupling (ie, physical binding) of the antibody with a detectable substance. Examples of detectable substances include various enzymes, prostate groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase or acetylcholinesterase; Examples of suitable prostate group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; An example of a luminescent material includes luminol; Examples of bioluminescent materials include luciferase, luciferin and aequorin and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

The antibody conjugates of the invention can be used to modify a given biological response. The therapeutic fraction should not be considered as limited to classical chemical therapeutic agents. For example, the pharmacological fraction may be a protein or a polypeptide that has a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or an active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; a protein such as tumor necrosis factor or interferon-gamma; or biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), colony stimulating factor of granulocytes and macrophages ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF") or other cytokines or growth factors.

Techniques for conjugating such therapeutic fractions with antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243 56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623 53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review1", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475 506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303 16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119 58 (1982).

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VII. Pharmaceutical compositions

In another aspect, the present invention provides a composition, for example, a pharmaceutical composition, which contains one or a combination of the monoclonal antibodies, or one or more antigen binding parts thereof (such as antigen binding fragments) , of the present invention, formulated together with a pharmaceutically acceptable carrier. In one embodiment, the compositions include a combination of multiple isolated human antibodies (eg, two or more) of the invention. Preferably, each of the antibodies in the composition binds to a different epitope previously selected from PD-L1.

The pharmaceutical compositions of the invention can also be administered in combination therapy, that is, in combination with other agents. For example, combination therapy may include a composition of the present invention with at least one or more additional therapeutic agents, such as anti-inflammatory agents, DMARD (disease-modifying anti-rheumatic drugs), immunosuppressive agents, chemotherapeutic agents and agents. of psoriasis The pharmaceutical compositions of the invention can also be administered together with radiotherapy. Co-administration with other antibodies, such as CD4 specific antibodies and IL-2 specific antibodies, is also included in the invention.

As used herein, "pharmaceutically acceptable carrier" includes each and every solvent, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders and the like that are physiologically compatible. Preferably, the vehicle is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (for example, by injection or infusion) Depending on the route of administration, the active compound, that is, antibody, bispecific and multispecific molecule, can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.

A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not confer any unwanted toxic effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1 19) Examples of such salts include acid addition salts and base addition salts.The acid addition salts include those from non-toxic inorganic acids, such as hydrochloric, nitric, phosphoric acid, sulfuric, bromhydric, iodhydric, phosphorous and the like, as well as non-toxic organic acids such as mono- and dicarboxylic aliphatic acids, substituted phenyl alkanoic acids, alkanoic hydroxy acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. bases include those from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as non-toxic organic amines, such as N, N'-dibenzylethylenediamine, N -methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by various methods known in the art. As one skilled in the art will appreciate, the route and / or mode of administration will vary depending on the desired results. The active compounds can be prepared with transporters that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Those skilled in the art generally know many methods for preparing such formulations or these are patented. See, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer with, a material that prevents its inactivation. For example, the compound can be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline solutions and aqueous buffers. Liposomes include water-in-water-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional means or agent is incompatible with the active compound, the use thereof in the pharmaceutical compositions of the invention is contemplated. Also active complementary compound compositions can be incorporated into the compositions.

The therapeutic compositions should normally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome or other suitable ordered structure with a high concentration of drug. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size.

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in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, monostearate and gelatin salts.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients listed above, as required, followed by microfiltration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle containing a basic dispersion medium and the other necessary ingredients as indicated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and cryo-drying (lyophilization) which produces a powder of the active principle plus any additional desired principle of a solution previously sterilized by filtration thereof. .

The dosage regimens are adjusted to provide the optimal desired response (for example, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the therapeutic situation. For example, the human antibodies of the invention can be administered once or twice a week by subcutaneous injection or once or twice a month by subcutaneous injection. It is especially advantageous to formulate the parenteral compositions in a unit dosage form to facilitate administration and uniformity of the dosage. As used herein, unit dosage form refers to physically suitable separate units as unit dosages for the subjects to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification of the unit dosage forms of the invention are dictated by and directly depend on (a) the exclusive characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the intrinsic limitations in the technique of compound formation. such as an active compound for the treatment of sensitivity in individuals.

In one embodiment, an agent of the invention is an antibody. As defined herein, a therapeutically effective amount of antibody (ie, an effective dosage) ranges from about 0.001 to 30 mg / kg of body weight, or from about 0.01 to 25 mg / kg of body weight, or of about 0.1 to 20 mg / kg body weight, or about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 at 6 mg / kg body weight. The person skilled in the art will appreciate that certain factors can influence the dosage necessary to effectively treat a subject, including but not limited to, the severity of the disease or disorder, prior treatments, general health and / or age of the subject. and other diseases present. Additionally, treatment of a subject with a therapeutically effective amount of an antibody may include a single treatment or, preferably, may include a series of treatments. It will also be appreciated that the effective dosage of antibody used for the treatment may increase or decrease throughout the cycle of a particular treatment. Dosage changes may result from the results of diagnostic tests.

Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like

For therapeutic compositions, the formulations of the present invention include those that are suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can be conveniently presented in a unit dosage form and can be prepared by any method known in the pharmacy art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject to be treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, from one hundred percent, this amount will vary from about 0.001 percent to about ninety percent active ingredient, as an alternative from about 0.005 percent to about 70 percent, or as an alternative from about 0.01 percent to about 30 percent.

The formulations of the present invention that are suitable for vaginal administration also include pessaries, buffers, creams, gels, pastes, foams or spray formulations containing said carriers as are known in the art that are appropriate. Dosage forms for topical or transdermal administration of the compositions of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservative, buffer or

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propellant that is required.

The terms "parenteral administration" and "parenterally administered" as linked herein mean modes of administration other than enteral and topical administration, usually by injection and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal administration, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intarespinal, epidural and intrasternal injection and infusion.

Examples of suitable aqueous and non-aqueous transporters that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils, such as olive, and injectable organic esters, such as ethyl oleate. Adequate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by using surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the presence of microorganisms can be guaranteed both by sterilization procedures, cited above, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, sorbic phenol acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. Additionally, prolonged absorption of the injectable pharmaceutical form can be carried out by including agents that delay absorption such as aluminum monostearate and gelatin.

When the compounds of the present invention are administered as pharmaceutical compounds to humans and animals, they can be administered alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (for example, 0.005 to 70%, such as from 0.01 to 30%) of active ingredient in combination with a pharmaceutically acceptable vehicle.

Regardless of the route of administration selected, the compounds of the present invention that can be used in suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. technique.

The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be modified to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. The dosage level selected will depend on various pharmacokinetic factors including the activity of the particular compositions of the present invention employed or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound. to be used, the duration of treatment, other factors, compounds and / or materials used in combination with the particular compositions used, age, sex, weight, condition, general health of the patient and previous medical history of the patient to be treated and similar factors well known to the medical technique. A doctor or veterinarian who has a habitual skill in the art can easily determine and prescribe the effective amount of the necessary pharmaceutical composition. For example, the doctor or the veterinarian can start with doses of the compounds of the invention used in the pharmaceutical compositions at lower levels than those necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound that is the lowest effective dose to produce a therapeutic effect. Such effective dose will generally depend on factors described above. It is preferred that the administration be intravenous, intramuscular, intraperitoneal or subcutaneous, preferably administered proximal to the target site. If desired, the effective daily dose of a therapeutic composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. When this is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

The therapeutic compositions can be administered with medical devices known in the art. For example, in one embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of well-known implants and modules useful in the present invention include: US Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medication at a controlled rate; United States Patent No. 4,486,194, which discloses a therapeutic device for administering medications through the skin; US Patent No. 4,448,233, which discloses a medication infusion pump for the delivery of medication at an exact infusion rate; US Patent No. 4,447,224, which discloses an implantable infusion apparatus of variable flow for continuous drug administration; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system that has

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multi-chamber compartments; and United States Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), these can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, US Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise one or more fractions that are selectively transported in specific cells or organs, thereby enhancing the administration of the targeted drug (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Exemplary addressing fractions include folate or biotin (see, for example, United States Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the inventions, as well as components of the molecules of the invention; p120 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett. 346: 123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4: 273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; In another embodiment, the liposomes include a targeting fraction. In yet another embodiment, the therapeutic compounds in the liposomes are administered by bolus injection to a site near the tumor or infection. The compositions should be fluid to the point that they are easily injectable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The composition must be sterile and fluid to the extent that the composition can be administered by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the necessary particle size in the case of dispersion and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

When the active compound is adequately protected, as described above, the compound can be administered orally, for example, with an inert diluent or an assimilable edible carrier.

VIII. Use and methods of the invention

The antibodies described herein (including derivatives and conjugates of the antibodies) and compositions containing the antibodies can be used in various diagnostic and therapeutic applications in vitro and in vivo (for example, positively or negatively modulating the immune response). For example, binding of the PD-1 ligand to PD-1 or B7-1 transmits an inhibitory signal. Thus, modulation of the interaction between PD-1 and a PD-1 ligand, or between a PD-1 ligand and a B7 polypeptide, results in the modulation of the immune response. PD-1 ligands also costimulate T lymphocytes. Thus, in one embodiment, antibodies that block the interaction between a PD-1 and PD-1 or B7 ligand can prevent inhibitory signaling. In one embodiment, antibodies that block the costimulatory signal of the PD-1 ligand block a costimulatory signal against an immune cell. Additionally, ligation of PD-L2 can induce cytokine secretion and survival of dendritic cells. Thus, antibodies that block the binding of PD-L2 can inhibit the survival of dendritic cells and reduce the expression of cytokines by dendritic cells, and through these mechanisms inhibit an immune response. In particular, the antibodies described herein are useful for diagnostic, prognostic, prevention and therapeutic applications related to particular conditions mediated by PD-1 and PD-L1, as discussed, for example, in Keir et al. (2008) Annu. Rev. Immunol. 26: 677; Sharpe et al., (2007) Nat. Immunol. 8: 239; Freeman et al. (2007) J. Exp. Med. 10: 2223.

Antibodies and antigen binding fragments of the present invention may be useful for diagnostic, prognostic, prevention and therapeutic applications with respect to neurodegenerative diseases (geriopsicosis, Alzheimer's disease, Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease , diabetic neuropathy, parkinsonian syndrome, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy and Creutzfeldt Creutzfeldt-Jakob disease).

Antibodies and antigen-binding fragments of the present invention may be useful for diagnostic, prognostic, prevention and therapeutic applications (such as the treatment and delay of the onset or progression of diseases) for diseases that accelerate the immune reaction, by example, asthma, autoimmune diseases (glomerular nephritis, arthritis, dilated cardiomyopathy-type disease, ulcerative colitis, syndrome

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of Sjogren, Crohn's disease, systemic erythematosis, chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, pachydermia, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulin-dependent diabetes mellitus, Behcet disease, Hashimoto's disease of Addison, dermatomyositis, myasthenia gravis, Reiter's syndrome, Graves' disease, pernicious anemia, Goodpasture's syndrome, sterility disease, chronic active hepatitis, penfigo, autoimmune thrombocytopenic purpura and autoimmune hemolytic anemia, chronic chronic hepatitis, Addison's disease anti-phospholipid, atopic allergy, autoimmune atopic gastritis, autoimmune achlorhydra, celiac disease, Cushing's syndrome, dermatomyositis, discoid lupus, erythematosis, Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia ambert-Eaton, lupoid hepatitis, some cases of lymphopenia, mixed connective tissue disease, pemphigoid, penfigo vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis nodosa, polyglandular autosindromes, primary biliary cirrhosis, primary sclerosing cholangitis, Raynaud's syndrome, recidive polychondritis Schmidt syndrome, limited scleroderma (or crest syndrome), sympathetic ophthalmia, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis, thyrotoxicosis, insulin resistance type b, ulcerative colitis and Wegener granulomatosis).

In one embodiment, the antibodies and antigen binding fragments of the present invention are useful for diagnostic, prognostic, prevention and therapeutic applications (such as treatment and delay of the onset or progression of diseases) for therapy and / or prevention of persistent infectious disease (for example, diseases from viral infections including HPV, HBV, hepatitis C virus (HCV), retroviruses such as human immunodeficiency virus (HlV-1 and HIV-2), herpes virus such as Epstein Barr (EBV), cytomegalovirus (CMV), HSV-1 and HSV-2 and influenza viruses Other antigens associated with pathogens that can be used as described herein are antigens of various parasites, including malaria, preferably peptide of malaria based on NANP repetitions.In addition, it includes bacterial, fungal and other pathogenic diseases such as Aspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofil aria, Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibriocholerae. Exemplary species include Neisseria gonorrhea, Mycobacterium tuberculosis, Candida albicans, Candida tropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema abortreum, Treponema brullum, Treponema brullum, Treponema abortreum, Treponema brullum, Treponema abortreum, Treponema brullum, Treponema brullum, Treponema abortreum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema brullum, Treponema abortreum, Treponema brullum, Treponema abortreum. Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichia coli, Actinobacmonusus abortiususus, Aboriginusus, Pellonae , Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillus fumigatus, Rough absidia, Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridium botulinum; or fungi, such as, for example, Paracoccidioides brasiliensis; or other pathogens, for example, Plasmodium falciparum. Priority pathogens of the National Institute of Allergy and Infectious Diseases (NIAID) are also included. These include, Category A agents, such as variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularaemia), filovirus (Ebola hemorrhagic fever, fever Marburg hemorrhage), arenavirus (Lassa (Lassa fever), Junin (Argentine hemorrhagic fever) and related viruses); Category B agents, such as Coxiella burnetti (Q fever), Brucella species (brucellosis), Burkholderia mallei (muermo), alphavirus (Venezuelan encephalomyelitis, eastern and western equine encephalomyelitis), Ricinus communis ricin toxin (castor seeds) , Clostridium perfringens epsilon toxin; Staphylococcus B enterotoxin B, Salmonella species, Shigella dysenteriae, Escherichia coli strain O157: H7, Vibrio cholerae, Cryptosporidium parvum; Category C agents, such as nipah virus, hantavirus, tick-borne hemorrhagic fiber virus, tick-borne encephalitis virus, yellow fever and multi-drug resistant tuberculosis; helminths, such as Schistosoma and Taenia; and protozoa, such as Leishmania (for example, L. mexicana) and Plasmodium.

In another embodiment, the antibodies or antigen binding fragments of the present invention are useful for diagnostic, prognostic, prevention and therapeutic applications for organ graft rejection, host graft disease (GVHD), allergic diseases and diseases caused. by attenuation of the immune reaction, in which PD-1, PD-L1 and / or PD-L2 participates, for example, cancer and infectious diseases.

The antibodies or antigen binding fragments described herein are administered to a subject according to known methods such as intravenous administration routes (eg, as a stroke or by continuous infusion over a period of time), subcutaneous, intramuscular , intraperitoneal, intracerebroespinal, intra-articular, intrasinovial, intrathecal or inhalation).

A subject is treated if one or more beneficial or desired results are obtained, including desirably clinical results. For the purposes of the present invention, the beneficial or desired clinical results include, but are not limited to, one or more of the following: decrease in one or more symptoms resulting from the disease, increase in the quality of life of people suffering from the disease. disease, decreased dose of other medications necessary to treat the disease, delayed disease progression and / or prolonged survival of individuals.

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1. Exploration methods

One aspect of the present disclosure relates to methods of using antibodies of the present disclosure to modulate an immune response by modulating costimulation (such as antibodies that modulate the function of PD-1, PD-L1 or PD-L2). These methods use screening tests, including cell-based and non-cell-based tests. In one embodiment the assays provide a method to identify antibodies that mature the interaction of a PD-L1 and PD-1. In another embodiment, the assays provide a method to identify antibodies that modulate the interaction between a PD-1 ligand and a B7 polypeptide.

In one embodiment, the disclosure refers to assays to explore candidates or test antibodies that bind to, or modulate the activity of PD-1, PD-L1 or PD-L2, for example, modulate the ability of the polypeptide to interact with (for example, join) your partner afin union. In one embodiment, a method of identifying an antibody to modulate an immune response involves determining the ability of the antibody to modulate, for example, potentiating or inhibiting, the interaction between PD-1 and a PD-1 ligand, and further determining the ability of the antibody to modulate the interaction between a PD-1 ligand and a B7 polypeptide. In one embodiment, an antibody that modulates the interaction between the PD-1 and PD-1 ligand (for example, without modulating the interaction between PD-1 ligand and the B7 polypeptide is selected). In another embodiment, an antibody that modulates the interaction between a PD-1 ligand and a B7 polypeptide (for example, without modulating the interaction between the PD-1 and PD-1 ligand) is selected.

In another embodiment, a method of identifying an antibody to decrease an immune response involves determining the ability of a candidate antibody to enhance the interaction between a PD-1 ligand and a B7 polypeptide and selecting an antibody that inhibits the interaction between the PD- ligand. 1 and polypeptide B7. In another embodiment, a method of identifying an antibody to decrease an immune response involves determining the ability of the candidate antibody to enhance the interaction between a PD-1 and PD-1 ligand and selecting an antibody that enhances the interaction between the PD-1 ligand. and PD-1.

In another embodiment, a cell-based assay, comprising contacting a cell that expresses PD-1, PD-L1 or PD-L2, with a test antibody and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the binding of PD-1 or the target PD-1 ligand with its binding partner. The determination of the ability of PD-1, ligand of PD-1 or B7 polypeptide to bind to, or interact with, its binding partner can be performed, for example, by measuring direct binding or by measuring an immune cell activation parameter.

For example, in a direct binding assay, PD-1 or the PD-1 ligand protein (or their respective target polypeptides) can be coupled with a radioisotope or enzymatic label such that the binding of PD-1 ligand to PD-1 or to polypeptide B7 can be determined by detecting the labeled protein in a complex. For example, PD-1 or PD-L1 can be labeled with 125I, 35S, 14C or 3H, directly or indirectly, and the radioisotope detected by direct broadcast or by scintillation counting. Alternatively, PD-1 or the PD-1 ligand can be enzymatically labeled for example with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme marker can be detected by determining the conversion of an appropriate substrate to the product.

It is also within the scope of this disclosure to determine the ability of a compound to modulate the interaction between PD-1 and a PD-1 ligand or between a PD-1 ligand and a B7 polypeptide without labeling any of the compounds that interact . For example, a microfisiometer can be used to detect the interaction of PD-1 and a PD-1 ligand or between a PD-1 ligand and a B7 polypeptide, with its target polypeptide, without marking any of PD-1, the PD ligand -1, B7 polypeptide, or the target polypeptide (McConnell, HM et al. (1992) Science 257: 1906-1912). As used herein, a "microfisiometer" (eg, Citosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-directed potentiometric sensor (LAPS). Changes in this rate Acidification can be used as an indicator of the interaction between a compound and a receptor.

In another embodiment, the determination of the ability of the antibody to antagonize the interaction between a given set of polypeptides can be performed by determining the activity of one more members of the set of polypeptides. For example, the activity of PD-1 or a PD-1 ligand can be determined by detecting the induction of a second cellular messenger (for example, tyrosine kinase activity), detecting the catalytic / enzymatic activity of an appropriate substrate, detecting the induction of an indicator gene (comprising a target sensitive regulatory element operably linked to a nucleic acid encoding a detectable label, for example, chloramphenicol acetyl transferase), or by detecting a cellular response regulated by PD-1 or the PD-1 ligand. The determination of the ability of the antibody to bind to or interact with said polypeptide can be performed, for example, by measuring the ability of a compound to modulate immune cell costimulation or inhibition in a proliferation assay, or by interference with the ability of said polypeptide to bind to antibodies that recognize a part thereof.

Antibodies that block or inhibit the interaction of a PD-1 ligand with a costimulatory receptor, as well as antibodies that promote a PD-1 ligand-mediated inhibitor signal can be identified by their

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ability to inhibit immune cell proliferation and / or effector function, or induce anergy when added to an in vitro assay. For example, cells can be cultured in the presence of an agent that stimulates signal transduction by means of an activation receptor. Various recognized cell activation readings can be used to measure cell proliferation or effector function (eg, antibody production, cytokine production, phagocytosis) in the presence of the activating agent. The ability of an assay antibody to block this activation can be readily determined by measuring the ability of the antibody to affect a decrease in proliferation or effector function that is measured using techniques known in the art.

For example, the antibodies of the present invention can be tested for the ability to inhibit or enhance costimulation in a T lymphocyte assay, as described in Freeman et al. (2000) J. Exp. Med. 192: 1027 and Latchman et al. (2001) Nat. Immunol. 2: 261. CD4 + T lymphocytes can be isolated from human PBMc and stimulated with anti-CD3 activation antibody. The proliferation of T lymphocytes can be measured by incorporation of thymidine 3H. An assay with or without costimulation of CD28 can be performed in the assay. Similar assays can be performed with Jurkat T lymphocyte cells and PHA blasts from PBMC.

In another additional embodiment, an assay of the present disclosure is an acellular assay in which PD-1 or a PD-1 ligand or a biologically active part thereof is contacted with a test antibody, and the ability of the test antibody to bind to the polypeptide, or biologically active part thereof. The binding of the antibody in assay with the PD-1 polypeptide or PD-1 ligand can be determined directly or indirectly as described above. In yet another embodiment, the assay includes contacting the polypeptide, or biologically active part thereof, with its binding partner to form a test mixture, contacting the test mixture with a test antibody and determining the ability of the test compound for interacting with the polypeptide in the test mixture, wherein determining the ability of the test antibody to interact with the polypeptide comprises determining the ability of the test antibody to preferentially bind to the polypeptide or biologically active part thereof. , in comparison with the union partner.

For example, a PD-1 ligand and a PD-1 polypeptide can be used to form an assay mixture and the ability of an assay antibody to block this interaction can be tested by determining the ability of PD-1 to bind to the PD-1 ligand. and determine the ability of the PD-1 ligand to bind to the PD-1 polypeptide, by one of the methods described above to determine binding. The determination of the ability of a PD-1 polypeptide to bind to a PD-1 ligand and to determine the ability of a PD-1 ligand to bind to a B7 polypeptide can also be performed using a technology such as real-time biomolecular interaction analysis. (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5: 699-705). As used herein, "BIA" is a technology for studying biospecific interactions in real time, without marking any of the interacting compounds (for example, BIAcore). Changes in the optical phenomenon of surface plasmon resonance can be used ( SPR) as an indication of real-time reactions between biological polypeptides PD-1, the PD-1 ligand and B7 polypeptide can be mobilized in a BIAcore microplate and the antibodies can be tested for binding to PD-1, the PD- ligand 1 and polypeptide B7 An example of the use of BIA technology is described by Fitz et al. (1997) Oncogene 15: 613.

The acellular assays of the present disclosure are directed to the use of soluble and / or membrane bound protein forms (for example, a PD-1 ligand or PD-1 proteins or biologically active parts thereof, or binding partners thereof). which binds a PD-1 or PD-1 ligand). In the case of acellular assays in which a protein that forms a membrane junction is used (eg, a PD-1 cell surface PD-1 ligand receptor) it may be desirable to use a solubilizing agent such that the form Membrane-bound protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit® , Isotridecipoil (ethylene glycol ether) n, 3 - [(3-

colamidopropyl) dimethylamino] -1-propane sulfonate (CHAPS), 3 - [(3-colamidopropyl) diethylamino] -2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl = N, N-dimethyl-3-ammonium -1-propane sulfonate.

In one or more embodiments of the test methods described above, it may be desirable to immobilize either PD-1, a PD-1 ligand and a B7 polypeptide, or an appropriate target polypeptide to facilitate separation of the complexing forms of the They do not form complex of one or both proteins, as well as accommodate the automation of the assay. The binding of a test antibody or a PD-1 ligand can be performed in any suitable container to contain the reactants. Examples of such containers include microtiter plates, test tubes and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to bind to a matrix. For example, glutathione-S-transferase / PD-1 fusion proteins, PD-1 ligand or B7 polypeptide or glutathione-S-transferase / target fusion proteins, can be absorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis , MO) or microtiter plates derivatized with glutathione which are then combined with the test compound, and the mixture is incubated under conditions that lead to the formation of the complex (for example, physiological conditions for salt and pH). After incubation, the beads or wells of the microtiter plate are washed to remove any unbound component, the matrix immobilized in the case of beads, determined the complex either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated

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of the matrix, and the level of binding or activity of PD-1, ligand of PD-1 or B7 polypeptide determined using conventional techniques.

In an alternative embodiment, the determination of the ability of the test compound to modulate the activity of PD-1 or PD-1 ligand can be performed by determining the ability of the test antibody to modulate the activity of a polypeptide that acts downstream of PD -1 or PD-1 ligand, for example, a polypeptide that interacts with the PD-1 ligand, or a polypeptide that operates downstream of PD-1, for example, interacting with the cytoplasmic domain of PD-1. For example, levels of second messengers can be determined, the activity of the polypeptide that interacts on an appropriate target can be determined, or the binding of the compound that interacts with an appropriate target can be determined as described above.

The present disclosure also relates to new antibodies identified by the screening assays described above. Accordingly, it is within the scope of the present invention to additionally use an antibody identified as described herein in an appropriate animal model. For example, an antibody identified as described herein can be used in an animal model to determine the efficacy, toxicity or side effects of treatment with said antibody. Alternatively, an antibody identified as described herein can be used in an animal model to determine the mechanism of action of said antibody. Additionally, the present invention relates to uses of new antibodies identified by the screening assays described above for treatments as described herein.

2. Prophylactic methods

The antibodies of the present invention and the disclosure can be used to prevent in a subject, a disease or condition associated with an unwanted, or undesirable immune response. Subjects who are at risk of a disease that could benefit from treatment with the antibodies or methods claimed can be identified, for example, by any or a combination of diagnostic or prognostic tests known in the art. The administration of a prophylactic antibody may occur before the manifestation of the symptoms associated with an unwanted or undesirable immune response. The appropriate antibody used for treatment can be determined based on clinical indications and can be identified using, for example, screening assays described herein.

3. Therapeutic methods

The antibodies of the present invention and disclosure can be used in therapeutic methods of modulating an immune response, for example, by modulating the interaction between PD-1 and a PD-1 ligand and / or a PD-1 ligand and a B7 polypeptide . For example, modulating the interaction between PD-1 and a PD-1 ligand, or between a PD-1 ligand and a B7 polypeptide, results in the modulation of the immune response. Thus, in one embodiment, antibodies that block the interaction between PD-1 and the PD-1 ligand can prevent inhibitory signaling. The PD-1 ligands can also potentiate costimulatory signals in T lymphocytes. Thus, in another embodiment, the antibodies that prevent the PD-L1 ligand from providing a costimulatory signal that can inhibit the costimulation of T lymphocytes.

These modulating antibodies can be administered in vitro (for example, by contacting the cell with an antibody) or, alternatively, in vivo (for example, by administering the agent to a subject). As such, the antibodies of the present invention and disclosure can be used in methods of treating an individual suffering from a disease or disorder that could benefit from modulating an immune response, for example, by modulating the interaction between a PD-1 ligand. and PD-1 or a B7 polypeptide. The invention provides antibodies and fragments thereof for use in treatment methods, as defined in the claims.

4. Negative regulation of immune responses

There are numerous embodiments of the invention as defined in the claims for positive regulation of the inhibitory function or negative regulation of the costimulatory function of a PD-1 ligand to thereby negatively regulate immune responses.

Negative regulation may be in the form of inhibition or blocking of an immune response already in progress or may involve the prevention of induction of an immune response. The functions of activated immune cells can be inhibited by negatively regulating immune cell responses, or by inducing specific anergy in immune cells, or both.

For example, the immune response can be negatively modulated using: anti-PD-L1 antibodies that block costimulation by PD-L1 (for example, even if it does not affect or increase the interaction between PD-L1 and PD-1) or that promotes binding of PD-L1 with PD-1 (for example, although it does not affect or although it inhibits costimulation by PD-L1).

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In one embodiment of the invention, tolerance is induced against specific antigens by co-administering an antigen with an antibody that blocks the costimulation of PD-L1. For example, tolerance can be induced in specific proteins. In one embodiment, immune responses can be inhibited against allergens, or against exogenous proteins in which an immune response is not desirable. For example, patients receiving factor VIII frequently generate antibodies against this coagulation factor. Co-administration of an antibody that blocks a costimulatory signal mediated by PD-L1 in combination with recombinant factor VIII (or by physically linked to Factor VIII, for example, by reticulation) may result in negative modulation.

In one embodiment, two different agents that negatively modulate immune responses can be combined as a single composition or administered individually (simultaneously or sequentially) to more negatively regulate immune responses mediated by immune cells in a subject. Additionally, a therapeutically active amount of one or more of the subject's antibodies may be used in conjunction with other negative modulation reagents to influence immune responses. Examples of other immunomodulatory reagents include, but are not limited to, antibodies that block a costimulatory signal, (for example, against CD28 or ICOS), antibodies that act as CTLA4 agonists, and / or antibodies against other immune cell markers (for example , against CD40, against the CD40 ligand, or against cytokines), fusion proteins (for example, CTLA4-Fc), and immunosuppressive drugs, (for example, rapamycin, cyclosporin A or FK506).

The negative regulation or prevention of a costimulation of the PD-1 ligand, or the promotion of an interaction between a PD-1 and PD-1 ligand is useful for negatively regulating the immune response, for example, in tissue transplantation situations, skin or organ in graft versus host disease (GVHD), or in inflammatory diseases such as systemic lupus erythematosus, and multiple sclerosis. For example, blocking the function of immune cells results in reduced tissue destruction in tissue transplantation. Normally, in tissue transplants, transplant rejection begins through its recognition as foreign by immune cells, followed by an immune interaction that destroys the transplant. The administration of an antibody that inhibits the costimulation of the PD-1 ligand alone or together with another negative modulating agent, before or at the time of transplantation can promote the generation of an inhibitory signal. In addition, inhibition of costimulatory signals of the PD-1 ligand or promotion of a PD-1 ligand or PD-1 inhibitory signals may also be sufficient to energize immune cells, thereby inducing tolerance in a subject. Prolonged tolerance induction by blocking a costimulatory signal mediated by PD-1 ligand may prevent the need for repeated administration of these blocking reagents.

To achieve sufficient immunosuppression or tolerance in a subject, it may also be desirable to block the costimulatory function of other polypeptides. For example, it may be desirable to block the function of B7-1, B7-2 or B7-1 and B7-2 by administering a soluble form of a combination of peptides that have activity on each of these antigens, blocking antibodies against these antigens or blocking small molecules (individually or together in a single composition) before or at the time of transplantation. Alternatively, it may be desirable to promote the inhibitory activity of a PD-1 or PD-1 ligand and inhibit a costimulatory activity of B7-1 and / or B7-2. Other negative modulating agents that can be used in conjunction with the negative modulation methods of the invention include, for example, agents that transmit an inhibitory signal by CTLA4, soluble forms of CTLA4, antibodies that activate an inhibitory signal by CTLA4, blocking antibodies against others. Immune cell markers or soluble forms of other pairs of receptor ligand (for example, agents that alter the interaction between CD40 and CD40 ligand (eg, antibodies antiligand CD40)), antibodies against cytokines or immunosuppressive drugs.

Negative modulation of immune responses is also useful in the treatment of autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of immune cells that are reactive against the tissue itself and that promote the production of cytokines and autoantibodies involved in disease pathology. The prevention of activation of self-reactive immune cells can reduce or eliminate disease symptoms. The administration of reagents that block the costimulation of immune cells by altering interactions between PD-1 ligand and B7 polypeptides, or by promoting the interaction between PD-1 and PD-1 ligand, without modulating or, although the interaction between the PD-1 ligand and a B7 polypeptide, are useful for inhibiting the activation of immune cells and preventing the activation of autoantibodies or cytokines that may be involved in disease processes. Additionally, agents that promote an inhibitory function of a PD-1 or PD-1 ligand can induce specific tolerance of autoreactive immune cell antigens, which could lead to prolonged disease relief. The efficacy of reagents in the prevention or relief of autoimmune disorders can be determined using various well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRLI / pr / lpr mice or NZB hybrid mice, murine autoimmune collagen-induced arthritis, diabetes mellitus in NOD mice and BB rats and murine experimental myasthenia gravis (see, for example, Paul ed., Fundamental Immunology, Raven Press, New York, third edition 1993, chapter 30).

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The inhibition of the activation of immune cells is therapeutically useful in the treatment of allergic reactions and allergy, for example, by inhibiting the production of IgE. An antibody that promotes a PD-1 ligand or PD-1 inhibitory function can be administered to an allergic subject to inhibit allergic responses mediated by immune cells in the subject. Inhibition of the costimulation of the PD-1 ligand of immune cells or the stimulation of a PD-1 ligand or the PD-1 inhibitor pathway may be accompanied by exposure to the allergen together with appropriate MHC polypeptides. Allergic reactions can be systematic or local in nature, depending on the route of entry of the allergen and the pattern of IgE deposition in mast cells or basophils. Therefore, the inhibition of allergic responses mediated by immune cells locally or systematically by administration of an inhibitory form of an agent that inhibits the interaction of a PD-1 ligand with a costimulatory receptor, or an antibody that promotes an inhibitory function of a PD-1 or PD-1 ligand.

The inhibition of the activation of immune cells through the blockage of costimulation of the PD-1 ligand, or through the promotion of the interaction between a PD-1 and PD-1 ligand, can also be therapeutically important in viral cell infections. immune For example, in the acquired immunodeficiency syndrome (AIDS), viral replication is stimulated by activation of immune cells. Modulation of these interactions can result in the inhibition of viral replication and therefore improve the course of AIDS. Modulation of these interactions can also be useful in promoting pregnancy maintenance. The PD-1 ligand is normally expressed at high levels in placental trophoblasts, the layer of cells that forms the interface between the mother and the fetus and that can play a role in preventing maternal rejection of the fetus. Women at risk of spontaneous abortion (for example, those who have previously experienced an abortion or who have difficulty conceiving) due to the immunological rejection of the embryo or fetuses can be treated with agents that modulate these interactions.

Negative regulation of an immune response by modulating the costimulation of a PD-1 ligand or by modulating the PD-1 / PD-1 binding ligand may also be useful in the treatment of autoimmune attack of autologous tissues. For example, the PD-1 ligand is normally expressed at high levels in the heart and can protect it from autoimmune attack. This is evidenced by the fact that Balb / c PD-1 knockout mice have massive autoimmune attack in the heart with thrombosis. Therefore, conditions that are caused or aggravated by autoimmune attack (for example, in this example, heart disease, myocardial infarction or atherosclerosis) can be improved or increased by modulating these interactions. Therefore, it is within the scope of the invention to modulate conditions aggravated by autoimmune attack, such as autoimmune disorders (as well as conditions such as heart disease, myocardial infarction and atherosclerosis).

5. Positive regulation of immune responses

Also therapeutically useful is blocking the interaction of a PD-1 ligand with PD-1 or B7-1 as a means to positively regulate an immune response. The positive regulation of immune responses can be in the form of potentiating an existing immune response or triggering an initial immune response. For example, the potentiation of an immune response using the subject compositions and methods is useful in case of infections with microbes (eg, bacteria, viruses or parasites). In one embodiment, an antibody that blocks the interaction of PD-L1 with PD-1 is used to enhance the immune response. Said antibody (for example, a non-activating antibody that blocks the binding of PD-L1 with PD-1) is therapeutically useful in situations where positive regulation of responses mediated by cells and antibodies would be beneficial. Exemplary disorders include viral skin disorders, such as herpes or shingles, in which case said agent can be administered topically to the skin. In addition, systemic viral diseases such as influenza, common cold and encephalitis could be relieved by the systematic administration of such agents.

Alternatively, immune responses can be enhanced in an infected patient through an ex vivo strategy, for example, by subtracting the patient's immune cells, contacting the immune cells in vitro with an antibody that blocks the interaction of a PD-1 ligand. with PD-1 and reintroducing the immune cells stimulated in vitro to the patient.

In certain cases, it would be desirable to additionally administer other agents that positively regulate immune responses, for example, forms of other members of the B7 family that transduce signals by costimulatory receptors, to further increase the immune response.

An antibody that blocks the interaction of a PD-1 ligand with PD-1 or B7-1 can be used prophylactically in vaccines against various polypeptides (eg, polypeptides from pathogens). Immunity against a pathogen (for example, a virus) can be induced by vaccinating with a viral protein together with an antibody that blocks the interaction of a PD-1 ligand with PD-1 or B7-1 in an appropriate adjuvant.

In another embodiment, the positive regulation or enhancement of a function of an immune response, as described herein, is useful in inducing tumor immunity.

In another embodiment, the immune response can be stimulated by the methods described herein,

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in such a way that the pre-existing tolerance is exceeded. For example, immune responses against antigens against which a subject cannot create a significant immune response, for example, against an autologous antigen, such as tumor specific antigens, can be induced by administering an antibody that blocks the interaction of a PD-ligand. 1 with PD-1. In one embodiment, an autologous antigen, such as a tumor specific antigen, can be co-administered. In another embodiment, an immune response against an antigen (eg, an autologous antigen) can be stimulated to treat a neurological disorder. In another embodiment, the subject agents can be used as adjuvants to reinforce responses against exogenous antigens in the active immunization process.

In one embodiment, immune cells of a subject are obtained and cultured ex vivo in the presence of an antibody as described herein, to expand the population of immune cells and / or enhance the activation of immune cells. In a further embodiment the immune cells are then administered to a subject. Immune cells can be stimulated in vitro, for example, by providing immune cells with a primary activation signal and a costimulatory signal, as is known in the art. Various agents can be used to costimulate the proliferation of immune cells. In one embodiment, the immune cells are cultured ex vivo according to the method described in PCT Publication No. WO 94/29436. The costimulatory polypeptide can be soluble, bound to a cell membrane, or attached to a solid surface, such as a pearl.

Other embodiments of the present invention are described in the following Examples. The present invention is further illustrated by the following examples.

Examples

The examples described below describe the generation of monoclonal antibodies suitable for therapeutic purposes that target human PD-1, PD-L1 and PD-L2. The human anti-PD-1, PD-L1 and PD-L2 human compound antibodies were generated from human anti EH12.2H7, 29E.2A3 and 24F.10C12 mouse antibodies, respectively. The segments of the sequence of the human V region were acquired from the databases of unrelated human antibody sequences (germ line and non-germ line). Each selected sequence segment (as well as the joints between segments) was tested for the possibility of joining the MHC class II using union prediction algorithms. All final compound human antibody sequence variants were designed to prevent T-cell epitopes. Composite human antibody region V regions were generated using synthetic oligonucleotides encoding combinations of human sequence segments. These were then cloned into vectors containing human constant regions, and antibodies were produced and tested for binding to target antigens by competition ELISA. The anti PD-1 and anti PD-L2 antibodies disclosed in the examples are outside the scope of the invention as defined in the claims, but are retained for background information.

Example 1: Design of human antibody variable region sequences, composed

Structural models of mouse regions EH12.2H7, 29E.2A3 and 24F.10C12 V were produced using Swiss Pdb and analyzed to identify important "restriction" amino acids in mouse regions V that could be essential for binding properties of the antibodies. Only the remains contained in the CDR were considered to be important, including the remains of the CDR defined in the definitions of Kabat and Chothia.

From the previous analyzes, it was considered that the human forms composed of EH12.2H7, 29E.2A3 and 24F.10C12 could be created without a wide latitude of sequences outside the cDr but with a narrow menu of possible alternative remains within the CDR sequences. Preliminary analyzes indicated that the corresponding sequence segments of various human antibodies could be combined to create CDRs similar or identical to those of the mouse sequences. For regions outside and flanking CDRs, a wide selection of human sequence segments were identified as possible components of the new variable regions of compound human antibody.

Based on the previous analyzes, a large preliminary set of sequence segments was selected that could be used to create variants of the human antibody compound EH12.2H7, 29E.2A3 and 24F.10C12 and were analyzed using class MHC binding prediction algorithms II and BLAST search through a proprietary database of known antibody sequence related to T lymphocyte epitopes. Sequence segments in which potential MHC class II binding peptides were identified or significant successes were scored against the base. of known T cell epitope data were discarded. This produced a reduced set of segments, and combinations of these were analyzed again, as indicated above, to ensure that the joints between the segments did not contain possible T-cell epitopes. Then the selected segments were combined to produce sequences of the variable region of heavy and light chain for the synthesis. For the three antibodies, five heavy chains and four light chains were constructed with the sequences detailed below:

Antigen Composite VH Sequences Composite VK Sequences

PD-1 Figure 2 (AE) Figure 3 (AD)

PD-L1 Figure 4 (AE) Figure 5 (AD)

PD-L2 Figure 6 (AE) Figure 7 (AD)

The sequence segments used to produce these compound human antibody sequences are detailed in Tables 1,2 and 3 for antibodies against PD-1, PD-L1 and PD-L2, respectively.

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Table 1. Derivation of human sequence segments comprising human antibodies, _________________________________ anti-PD-1 compounds __________________________________

 Genbank Registration No.

 VH3 sequence (a)

 BAA75018

 QVQLVQSGHEVKQPGASVK (SEQ ID NO: 82)

 AAG00910

 MSCKASGYSFTS (SEQ ID NO: 83)

 AAY18543

 SGYSFTSSWI (SEQ ID NO: 84)

 AAY57105

 WIHWV (SEQ ID NO: 85)

 AAG00910

 KQ

 AAD16517

 QAPGQGLEWIG (SEQ ID NO: 86)

 AAD53797

 GLEWIGYIYPS (SEQ ID NO: 87)

 CAA08742

 STGF (SEQ ID NO: 88)

 CAC87219

 TEYN (SEQ ID NO: 89)

 AAT96419

 QKF

 AAA17939

 KDR

 AAR02530

 DRAT (SEQ ID NO: 90)

 AAA17939

 TLT

 AAM87977

 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91)

 CAA78534

 STAYMELSSLRSEDTAVYYCARWRD (SEQ ID NO: 92)

 AAV40096

 DSSGY (SEQ ID NO: 93)

 AAR38557

 Yha

 AAW29142

 AMD

 IGHJ4

 DYWGQGTLVTVSS (SEQ ID NO: 94)

 Genbank Registration No.

 VH4 (b)

 Sequence

 QVQLVQSGHEVKQPGASVK (SEQ ID NO: 82)

 BAA75018

 AAG00910

 MSCKASGYSFTS (SEQ ID NO: 83)

 AAY18543

 SGYSFTSSWI (SEQ ID NO: 84)

 AAA02616

 HWVRQAPGQGLEWIG (SEQ ID NO: 95)

 AAD53797

 GLEWIGYIYPS (SEQ ID NO: 87)

 CAA08742

 STGF (SEQ ID NO: 88)

 CAC87219

 TEYN (SEQ ID NO: 89)

 AAT96419

 QKF

 AAA17939

 KDR

 AAR02530

 DRAT (SEQ ID NO: 90)

 AAA17939

 TLT

 AAM87977

 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91)

 CAA78534

 STAYMELSSLRSEDTAVYYCARWRD (SEQ ID NO: 92)

 AAV40096

 DSSGY (SEQ ID NO: 93)

 AAR38557

 Yha

 AAW29142

 AMD

 IGHJ4

 DYWGQGTLVTVSS (SEQ ID NO: 94)

 Genbank Registration No.

 Vk3 sequence (c)

 AAY16615

 EIVLTQSPATLSLSPGQR (SEQ ID NO: 96)

 AAD09377

 RLTISCRASQ (SEQ ID NO: 97)

 AAA99362

 TISCRASQSVST (SEQ ID NO: 98)

 AAL04518

 SVSTSGYSYMHW (SEQ ID NO: 99)

 AAA58912

 WYQQKPDQSPKLLIK (SEQ ID NO: 100)

 AAD16648

 FGS

 AAD19478

 SNLESG (SEQ ID NO: 101)

 AAL10884

 GIPARFSGSGSGTDFTLTISSLEPEDFA (SEQ ID NO: 102)

 AAD16559

 PEDFATYYCQHS (SEQ ID NO: 103)

 AAA99326

 SW

 AAC16811

 EIP

 human J2

 YTFGQGTKT EIK (SEQ ID NO: 104)

 Genbank Registration No.

 Vk4 sequence (d)

 AAB53267

 DIVLTQSP (SEQ ID NO: 105)

 AAY16615

 IVLTQSPATLSLSPGQR (SEQ ID NO: 106)

 AAD09377

 RLTISCRASQ (SEQ ID NO: 97)

 AAA99362

 TISCRASQSVST (SEQ ID NO: 98)

 AAL04518

 SVSTSGYSYMHW (SEQ ID NO: 99)

 AAA58912

 WYQQKPDQSPKLLIK (SEQ ID NO: 100)

 AAD16648

 FGS

 AAD19478

 SNLESG (SEQ ID NO: 101)

 AAL10884

 GIPARFSGSGSGTDFTLTISSLEPEDFA (SEQ ID NO: 102)

 AAD16559

 PEDFATYYCQHS (SEQ ID NO: 103)

 AAA99326

 SW

 AAC16811

 EIP

 human J2

 YTFGQGTKLEIK (SEQ ID NO: 104)

Table 2. Derivation of human sequence segments comprising human antibodies _________________________________ anti-PD-L1 compounds __________________________________

 Genbank Registration No.

 VH2 sequence (a)

 ABI50688

 EVQLVQSGAEVKKPGASVK (SEQ ID NO: 107)

 AAG00910

 MSCKASGY (SEQ ID NO: 108)

 ABI50688

 SCKASGYTFTSY (SEQ ID NO: 109)

 AAC50839

 SYVMHWV (SEQ ID NO: 110)

 CAC43594

 WVKQ (SEQ ID NO: 111)

 AAA18267

 QAPGQRLEWIG (SEQ ID NO: 112)

 ABF20472

 GY

 AAD30737

 VNPF (SEQ ID NO: 113)

 CAL06274

 NDGT (SEQ ID NO: 114)

 CAC43212

 KYN

 CAC87219

 Yne

 CAD31770

 EM

 AAR32413

 FKGR (SEQ ID NO: 115)

 AAG30515

 GRAT (SEQ ID NO: 116)

 ABA62048

 TLT

 ABI50549

 TSD

 AAR32572

 DKSTSTAYMELSSLRSEDTAVYYCA (SEQ ID NO: 117)

 AAC18225

 AVYYCARQA (SEQ ID NO: 118)

 AAV39747

 AWGY (SEQ ID NO: 119)

 IGHJ5 * 02

 PWGQGTLVTVSS (SEQ ID NO: 120)

 Genbank Registration No.

 VH4 sequence (b)

 ABI50688

 EVQLVQSGAEVKKPGASVK (SEQ ID NO: 107)

 AAG00910

 MSCKASGY (SEQ ID NO: 108)

 ABI50688

 SCKASGYTFTSY (SEQ ID NO: 109)

 AAC50839

 SYVMHWV (SEQ ID NO: 110)

 AAA18267

 WVRQAPGQRLEWIG (SEQ ID NO: 121)

 ABF20472

 GY

 AAD30737

 VNPF (SEQ ID NO: 113)

 CAL06274

 NDGT (SEQ ID NO: 114)

 CAC43212

 KYN

 CAC87219

 Yne

 CAD31770

 EM

 AAR32413

 FKGR (SEQ ID NO: 115)

 AAG30515

 GRAT (SEQ ID NO: 116)

 ABA62048

 TLT

 ABI50549

 TSD

 AAR32572

 DKSTSTAYMELSSLRSEDTAVYYCA (SEQ ID NO: 117)

 AAC 18225

 AVYYCARQA (SEQ ID NO: 118) (b) _________________

 AAV39747

 AWGY (SEQ ID NO: 119)

 IGHJ5 * 02

 PWGQGTLVTVSS (SEQ ID NO: 120)

 ; Genbank Registration No.

 sequence Zk1 (c)

 CAA31193

 DIVLTQSPASLALS (SEQ ID NO: 122)

 ABA26115

 .SPGERAT (SEQ ID NO: 123)

 AAQ21828

 IS V

 CAA51101

 GO

 AAA58691

 YYGTSL (SEQ ID NO: 124)

 AAY33369

 VQWYQQKPGQ (SEQ ID NO: 125)

 ABI74051

 WYQQKPGQPPKLLIY (SEQ ID NO: 126)

 CAC39383

 PKLLIYAASS (SEQ ID NO: 127)

 CAA38592

 SVDS (SEQ ID NO: 128)

 AAK26833

 DSGVPSRFSGSGSGT (SEQ ID NO: 129)

 AAM46660

 RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130)

 AAL04518

 EEEDAA (SEQ ID NO: 131)

 AAK68016

 AMYFCQQ (SEQ ID NO: 132)

 CAK50767

 3R

 AAP23227

 RVPYTFG (SEQ ID NO: 133)

 Human J2

 YTFGQGTKLEIK (SEQ ID NO: 104)

 Genbank Registration No.

 Vk2 sequence (d)

 CAA31193

 DIVLTQSPASLALS (SEQ ID NO: 122)

 CAE54363

 IVLTQSPATLSLSPGE (SEQ ID NO: 134)

 ABA26115

 LSPGERAT (SEQ ID NO: 123)

 AAQ21828

 IS V

 CAA51101

 GO

 AAA58691

 YYGTSL (SEQ ID NO: 124)

 AAY33369

 VQWYQQKPGQ (SEQ ID NO: 125)

 ABI74051

 WYQQKPGQPPKLLIY (SEQ ID NO: 126)

 CAC39383

 PKLLIYAASS (SEQ ID NO: 127)

 CAA38592

 SVDS (SEQ ID NO: 128)

 AAK26833

 DSGVPSRFSGSGSGT (SEQ ID NO: 129)

 AAM46660

 RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130)

 AAA58912

 TINSLEAEDAA (SEQ ID NO: 135)

 AAK68016

 AMYFCQQ (SEQ ID NO: 132)

 CAK50767

 MR

 AAP23227

 RVPYTFG (SEQ ID NO: 133)

 Human J2

 YTFGQGTKLEIK (SEQ ID NO: 104)

 Genbank Registration No.

 Vk4 sequence (e)

 CAA31193

 DIVLTQSPASLALS (SEQ ID NO: 122)

 CAE54363

 IVLTQSPATLSLSPGE (SEQ ID NO: 134)

 ABA26115

 LSPGERAT (SEQ ID NO: 123)

 AAQ21828

 IS V

 CAA51101

 GO

 AAA58691

 YYGTSL (SEQ ID NO: 124)

 AAY33369

 VQWYQQKPGQ (SEQ ID NO: 125)

 ABI74051

 WYQQKPGQPPKLLIY (SEQ ID NO: 126)

 CAC39383

 PKLLIYAASS (SEQ ID NO: 127)

 CAA38592

 SVDS (SEQ ID NO: 128)

 AAK26833

 DSGVPSRFSGSGSGT (SEQ ID NO: 129)

 AAM46660

 RFSGSGSGTDFTLTINSLE (SEQ ID NO: 130)

 AAA58912

 TINSLEAEDAATYFC (SEQ ID NO: 136)

 AAK68016

 AMYFCQQ (SEQ ID NO: 132)

 CAK50767

 MR

 AAP23227

 RVPYTFG (SEQ ID NO: 133)

 Human J2

 YTFGQGTKLEIK (SEQ ID NO: 104)

Table 3. Derivation of human sequence segments comprising human anti-PD antibodies - _______________________________________L2 compounds_______________________________________

 Genbank Registration No.

 VH2 sequence (a)

 ABF83419

 QVQLVQSGAEVKKPGASVK (SEQ ID NO: 137)

 AAG00910

 MSCKASGY (SEQ ID NO: 108)

 ABF83419

 SCKASGYTFTGY (SEQ ID NO: 138)

 AAL17955

 TMHWV (SEQ ID NO: 139)

 CAC43594

 WVKQ (SEQ ID NO: 111)

 AAL17955

 QAPG (SEQ ID NO: 140)

 AAF40162

 GQGLEWIG (SEQ ID NO: 141)

 AAR02558

 GYINP (SEQ ID NO: 142)

 AAR32283

 INPRSG (SEQ ID NO: 143)

 AAR02553

 GYT

 CAC87219

 TEYN (SEQ ID NO: 89)

 AAT96419

 QKF

 AAA17939

 KDR

 AAB06403

 RTT

 AAA17939

 TLT

 AAG30529

 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91)

 ABE66740

 DTAVYYCARPW (SEQ ID NO: 144)

 ABK81281

 WFAYWGQGT (SEQ ID NO: 145)

 IGHJ4

 YWGQGTLVTVSS (SEQ ID NO: 146)

 Genbank Registration No.

 VH4 sequence (b)

 ABF83419

 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGY (SEQ ID NO: 147)

 AAL17955

 TMHWVRQAPG (SEQ ID NO: 148)

 AAF40162

 GQGLEWIG (SEQ ID NO: 141)

 AAR02558

 GYINP (SEQ ID NO: 142)

 AAR32283

 INPRSG (SEQ ID NO: 143)

 AAR02553

 GYT

 CAC87219

 TEYN (SEQ ID NO: 89)

 AAT96419

 QKF

 AAA17939

 KDR

 AAB06403

 RTT

 AAA17939

 TLT

 AAG30529

 TADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 91)

 ABE66740

 DTAVYYCARPW (SEQ ID NO: 144)

 ABK81281

 WFAYWGQGT (SEQ ID NO: 145)

 IGHJ4

 YWGQGTLVTVSS (SEQ ID NO: 146)

 Genbank Registration No.

 Vk2 sequence (c)

 AAD16249

 DIVMTQSP (SEQ ID NO: 149)

 CAA31193

 PASL (SEQ ID NO: 150)

 AAA58913

 LSVTPGEKVTITC (SEQ ID NO: 151)

 AAQ99244

 CKSSQSLL (SEQ ID NO: 152)

 ABA71421

 LNS

 AAD19451

 GN

 AAS86065

 QK

 AAD14073

 KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153)

 AAZ09126

 RFTGSGSGTDFTLTISSLQAEDVAVYYCQ (SEQ ID NO: 154)

 CAA31484

 NDY

 CAC87582

 YSYPL (SEQ ID NO: 155)

 human J1

 TFGQGTKLEIK (SEQ ID NO: 156)

 Genbank Registration No.

 Vk3 sequence (d)

 AAD16249

 DIVMTQSP (SEQ ID NO: 149)

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 AAA58913

 VMTQSPAFLSVTPGEKVTITC (SEQ ID NO: 157)

 AAQ99244

 CKSSQSLL (SEQ ID NO: 152)

 ABA71421

 LNS

 ADF 19451

 GN

 AAS86065

 QK

 AAD14073

 KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153)

 AAZ09126

 RFTGSGSGTDFTLTISSLQAEDVAVYYCQ (SEQ ID NO: 154)

 CAA31484

 NDY

 CAC87582

 YSYPL (SEQ ID NO: 155)

 human J1

 TFGQGTKLEIK (SEQ ID NO: 156)

 Genbank Registration No.

 Vk4 sequence (e)

 AAD 16249

 DIVMTQSP (SEQ ID NO: 149)

 AAA58913

 VMTQSPAFLSVTPGEKVTITC (SEQ ID NO: 157)

 AAQ99244

 CKSSQSLL (SEQ ID NO: 152)

 ABA71421

 LNS

 ADF 19451

 GN

 AAS86065

 QK

 AAD14073

 KNYLTWYQQKPGQPPKLLIYWASTRESGVPDRF (SEQ ID NO: 153)

 CAD44754

 RFSGSGSGTDFTLTISSLQAEDVAVYYCQ (SEQ ID NO: 158)

 CAA31484

 NDY

 CAC87582

 YSYPL (SEQ ID NO: 155)

 human J1

 TFGQGTKLEIK (SEQ ID NO: 156)

Example 2: generation and testing of human antibodies, compounds

The VH and VK region genes of the human antibody composed of initial variant 1, were synthesized for EH12.2H7, 29E.2A3 and 24F.10C12 using a series of overlapping oligonucleotides that hybridized, ligated and amplified by PCR to produce full length synthetic V regions (Figure 2A, Figure 3A, Figure 4A, Figure 5A, Figure 6A and Figure 7A). For each compound human antibody, subsequent sequence variants were constructed using long overlapping oligonucleotides and PCR using the initial variant 1 as template. The assembled variants were then cloned directly into expression vectors (Figure 1) and their sequences were verified.

All combinations of the chimeric and compound heavy and light chains (i.e. a total of 20 pairings for each antibody) were stably transfected into NS0 cells by electroporation and selected on media (high glucose DMEM with L-glutamine and Na pyruvate, 5% ultra low FCS IgG, pen / strep - all from Invitrogen) containing 200 nM methotrexate. Various drug resistant colonies were tested for each construct for expression levels and the best expression lines were selected and frozen with liquid nitrogen.

The supernatants of the lines that were best expressed from each combination were quantified using an Fc, Kappa light chain detection ELISA capture compared to an IgG1 / kappa pattern. Then the quantified supernatants were tested in a competition ELISA for binding with their target antigen. Ninety-six wells of Maxisorb ™ plates (Nunc) were coated overnight at 4 ° C with 50 µl / well of Fc-PD-1, Fc-PD-L1 or human Fc-PD-L2 1 jg / ml ( R&D Systems) in carbonate buffer pH 9.6. Duplicate titers of the mouse reference antibody and samples of compound human antibody (in the range of 0.0078 jg / ml to 8 jg / ml) were generated and mixed with a constant concentration (40 ng / ml) of reference of biotinylated mouse in PBS pH 7.4 / 2% BSA. The titers, 100 µl / well, were added to washed test plates (4x with PBS pH 7.4 / 0.05% Tween 20) and incubated at room temperature for 1 hour. The plates were washed as indicated above, and 100 µl / well of a 1/1000 dilution of streptavidin HRP (Sigma) in PBS pH 7.4 / 2% BSA was added and incubated for 1 more hour at room temperature . After further washing, the bound biotinylated reference antibody was detected with OPD substrate 100 µl / well. The absorbance at 490 nm was measured and the binding curves of the test antibodies were compared with the mouse reference standard. The absorbance was plotted against the sample concentration and the straight lines were adjusted across each of the data sets. The equations of the lines were used to calculate the concentration necessary to inhibit the binding of biotin-EH12.2H7 with PD-1, the binding of biotin-29E.2A3 with PD-L1 and the binding of biotin-24F.10C12 with PD -L2 50% human (IC50).

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The antibodies with the highest IC50 value were selected and the cell lines of all these variants of EH12.2H7, 29E.2A3 and 24F.10C12 antibodies were increased in volume to 100 ml and grown to saturation. The antibodies were purified from each culture by affinity chromatography with protein A. In summary, the pH of the supernatants was adjusted with a volume of 0.1 PBS 10 x pH 7.4 and passed on protein A columns of 1 ml Mab Select Sure (GE Healthcare). The columns were washed with 10 volumes of PBS pH 7.4 before elution with 50 mM citrate buffer pH 3.0. 1 ml fractions were collected and immediately neutralized with 0.1 ml of 1M Tris-HCl pH 9.0. Fractions containing protein (according to absorbance at 280 nm) were pooled, the buffer was changed in PBS pH 7.4 and the purified antibodies were stored at + 4 ° C. Figures 8A-C show a 1 | jg SDS-PAGE gel of each antibody, taken with coomassie blue. Antibody concentrations were calculated by UV absorption based on the molar extinction coefficients calculated such that Eo, i% at 280 nm = 1.61 for EH12.2H7, E0.1% at 280 nm = 1.46 for 29E.2A3 and E0.1% at 280 nm = 1.57 for 24F.10C12.

The purified antibodies were tested for binding to Fc-PD-1, Fc-PD-L1 or human PD-Fc-L2 by ELlSA competition assay as described above. Titrations of the test antibodies were made from 0.0625 jg / ml to 8.0 jg / ml in duplicate. The absorbance at 490 nm was measured and plotted against the concentration of the test antibody (Figures 9A-C, 10A-C).

Table 4 summarizes the results of the combinations of the VH and VK variant sequences of the compound for anti-PD-1, PD-L1 and PD-L2 antibodies. For EH12.2H7 all humanized antibodies had an IC50 value that is improved compared to the mouse reference, particularly VH4 / VK3 that had a two-fold increase in binding. In the case of 29E.2A3, the variants VH2 / VK1 and VH2 / VK4 had equivalent union with respect to the mouse reference while the variants VH2 / VK2 and VH4 / VK2 had a union reduced to 1.75 and 1.36 times respectively. For 24F.10C12, all selected variants had a similar, although slightly reduced, union compared to the mouse reference (1.13 times).

Table 4: IC50 values for human antibody sequence variants, compound PD-1, PD-L1 and PD-L2

 EH12.2H7 IC50 jg / ml antibody

 Antibody 29E.2A3 IC50 jg / ml Antibody 24F.10C12 CI50 jg / ml

 mouse

 1.23 mouse 0.28 mouse 0.52

 VH3 / VK3

 0.93 VH2 / VK1 0.27 VH2 / VK2 0.58

 VH3 / VK4

 0.74 VH2 / VK2 0.49 VH2 / VK3 0.59

 VH4 / VK3

 0.57 VH4 / VK2 0.38 VH4 / VK2 0.60

 VH4 / VK4

 0.91 VH2 / VK4 0.29 VH4 / VK4 0.58

As a result of these experiments, the human compound antibodies specific for human PD-1, PD-L1 and PD-L2 had been constructed from segments of amino acid sequences from unrelated regions of human antibody. All CDRs and framework regions conserved in the composite human antibody variants comprised more than one segment of unrelated human sequence (obtained from the human sequence database) and all compound human antibodies were specifically designed to prevent T lymphocyte epitopes Four major candidates were initially selected for binding to human PD-1, PD-L1 or PD-L2 and, after further analysis, it was shown that they had binding with a factor of two on the murine antibody.

Example 5: Enhanced stimulation of T lymphocyte activation by inhibition of interaction between PD-1: PD ligand

The PD-1 signaling pathway inhibits moderate costimulatory signals of TCR / CD28, reducing cytokine production first without a decrease in T lymphocyte proliferation. As the costimulatory signals of TCR / CD28 weaken, the PD- route 1 dominates, with a great reduction in cytokine production accompanied by a reduction in proliferation. Therefore, to confirm that inhibition of the PD-1 pathway by inhibiting interaction with PD-L1 or PD-L2 using the human antibodies composed of the invention potentiates the activation of T lymphocytes, mixed lymphocyte reactions are performed. (MLR).

Immature myeloid dendritic cells are isolated by culturing monocytes of human peripheral blood in IL-4 and GM-CSF. Exposure of immature dendritic cells to an inflammatory cocktail of IL-1 p, TNF-a, IL-6 and PGE2 causes the development of mature dendritic cells that function as APC. However, the addition of IL-10 to the inflammatory cytokines given during the ripening phase produces APCs that work only 1/6 to 1/3 as well.

T lymphocyte activation (MLR) assays were performed, using IL-10 treated dendritic cells as APC, in the presence of human antibodies, compounds against PD-1, PD-L1 and / or PD-L2, or control antibodies. The addition of anti-PD-1, anti-PD-L1 and / or PD-L2 mAbs to dendritic cell cultures treated with IL-1 or more allogenic T lymphocytes is predicted to produce an increase in T lymphocyte proliferation and expression of cytokines, compared to cultures treated with control IgG. A combination of anti-PD-1 antibodies with anti-antibody

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PD-L1, anti-PD-L2 antibodies, may also produce an increase in stimulation greater than that observed with any of the antibodies alone.

Example 6: Inhibition of the PD-1 pathway in chronically infected mice

Mice infected with various strains of lymphocytic choriomeningitis virus (LCMV) were used to study the effect of chronic viral infection on CD8 T lymphocyte function. The Armstrong strain of LCMV causes an acute infection that is eliminated after 8 days, leaving behind a long-lived population of resting memory CD8 T lymphocytes, very functional. On the other hand, the Cl-13 strain of LCMV establishes a persistent infection in the host, characterized by a viremia of up to three months.

To confirm that blocking PD-1 signaling restores T lymphocyte function and enhances viric control during chronic LCMV infection, PD-1 signaling is interrupted during chronic LCMV infection using anti-PD-1 antibodies, anti-PD antibodies. -PD-L1 and / or human anti-PD-L2 antibodies composed of the invention. The antibodies are administered every three days to mice infected with LCMV Cl-13 from day 23 to day 37 post-infection. It was expected that on day 37 there would be several more increases in CDM T lymphocytes specific for LCMV in treated mice with respect to untreated controls. It was also expected that the induction of proliferation would be specific against given CD8 T cells and the number of CD4 T cells in the spleen would probably be the same in both treated and untreated mice.

In addition to an increase in the proliferation of CD8 T lymphocytes, inhibition of PD-1 signaling was also expected to produce increased production of antiviral cytokines in virus-specific CD8 T lymphocytes. The production of IFN-gamma and TNF-alpha by CD8 T lymphocytes would possibly be several times more in treated mice compared to untreated ones. Viric elimination should also be accelerated, and reduced viral titres should be observed in lung and kidney on day 37 post-infection in treated mice, although untreated mice probably had significant levels of virus in all these tissues.

CD4 T lymphocytes play a major role in the generation and maintenance of CD8 T lymphocyte responses. In this sense, CD8 T lymphocytes sensitized in the absence of CD4 T lymphocytes cannot create normal immune responses, and therefore are often referred to as "defenseless T lymphocytes." Additionally, chronic infection of LCMV-Cl-13 is more severe in the absence of CD4 T lymphocytes. Consequently, defenseless T lymphocytes, generated during LCMV-Cl-13 infection, exhibit functional dysfunction even deeper than T lymphocytes generated in the presence of TCD4 lymphocytes.

CD4 T lymphocytes are depleted at the time of infection LCMV-Cl-13 and mice are treated with anti-PD-1 antibodies, anti-PD-L1 antibodies and / or human anti-PD-L1 antibodies composed of the present invention. from day 46 to day 60 post-infection. After treatment, it is expected that treated mice will have several times more CD8 T-lymphocytes specific to LCMV in their spleen than untreated control mice. This increase in virus-specific CD8 T lymphocytes in treated mice may produce an increase in proliferation, detected by incorporation of BrdU. BrdU analysis is performed by introducing 1 mg / ml BrdU into drinking water during treatment and staining is performed according to the manufacturer's protocol (BD Biosciences, San Diego, Calif.).

To confirm that inhibition of PD-1 signals increases the lithic activity of virus-specific CD8 T lymphocytes, depleted defenseless, the ex vivo lithic activity of virus-specific CD8 T lymphocytes is detected after treatment using a 51Cr release assay (Wherry et al., 2003. J. Virol. 77: 4911-27). Viral titres are expected to be reduced several times in the spleen, liver, lung and serum after two weeks of treatment with respect to untreated mice.

Example 7: Administration of a vaccine with a PD-1 signaling inhibitor.

A strategy to reinforce T lymphocyte responses during a persistent infection is therapeutic vaccination. The logic of this strategy is that endogenous antigens may not be present in an optimal or immunogenic manner during chronic viral infection and that the supply of antigens in the form of a vaccine may provide a more effective stimulus for specific T and B lymphocytes. virus. Using the chronic LCMV model, mice were given a recombinant vaccinia virus expressing the GP33 epitope of LCMV as a therapeutic vaccine (VVGP33), which produces a modest potentiation of CD8 T lymphocyte responses in some chronically infected mice. This therapeutic vaccination is combined with the anti-PD-1 antibodies, anti-PD-L1 antibodies and human anti-PD-L2 antibodies and compounds of the invention. LCMV-specific T lymphocyte responses are expected to be reinforced at a higher level compared to any treatment alone and that the effect of the combined treatment is likely to be much more than additive.

Example 8: Chimpances as a model of immunotherapy for persistent HCV infection.

Chimpanzees provide a model of HCV persistence in humans. Defects in immunity of T lymphocytes that lead to prolonged virus persistence including both a deficit in CD4 helper T lymphocytes

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HCV species as an activity of defective or altered CD8 T lymphocytes. Persistent infected chimpanzees are treated with anti-PD-1 antibodies, anti-PD-L1 antibodies and / or human anti-PD-L2 antibodies, compounds, of the invention. The efficacy of blocking inhibitory pathways was determined, combined with vaccination using recombinant structural and non-structural HCV proteins, and whether such strategies can enhance the frequency and longevity of virus-specific memory T lymphocytes. The defect in immunity of T lymphocytes is exclusively specific for HCV in persistently infected humans and chimpanzees. The antiviral activity can then be restored by providing the chimpanzees with humanized monoclonal antibodies that block signaling through these molecules.

Persistently infected chimpanzees were treated with anti-PD-1 antibodies, anti-PD-LI antibodies and / or human anti-PD-L2 antibodies, compounds of the invention. After treatment with the antibodies, humoral and cellular immune responses were determined, as well as the ACV RNA load. Samples were collected at weeks 1, 2, 3, 5 and 8, and then at monthly intervals. Samples include: 1) serum for analysis of transaminases, autoantibodies, neutralizing antibodies against HCV, and cytokine responses, 2) plasma for viral load and genome evolution, 3) PBMC for in vitro measures of immunity, expression and receptor function costimulator / inhibitor, 4) recent liver (not fixed) for isolation of intrahepatic lymphocytes and RNA, and 5) fixed liver (included in formalin / paraffin) for histological and immunohistochemical analyzes. Regional lymph nodes will also be collected in 2 or 3 times to assess the expression of co-inhibitory molecules and splicing variants using immunohistochemical and molecular techniques.

To determine whether a vaccination with HCV antigens enhances the therapeutic effect of the antibodies, chimpanzees were treated as follows: 1) intramuscular immunization with recombinant envelope glycoproteins E1 and E2 (in adjuvant MF59) and other proteins (nucleus plus NS 3 , 4, and 5 formulated with ISCOMS) weeks 0, 4 and 24; 2) intramuscular immunization with the vaccine used in, although co-administered with anti-PD-1 antibodies, anti-PD-L1 antibodies and / or human anti-PD-L2 antibodies, composed of the antibodies of the invention. HCV-specific B and T lymphocyte responses were monitored at monthly intervals after immunization for a period of one year.

The markers examined in total and positive HCV tetramer T lymphocytes in this analysis included differentiation markers (for example, CD45RA / RO, CD62L, CCR7 and CD27), activation (e.g., CD25, CD69, CD38 and HLA-DR), survival / proliferation (for example, bcl-2 and Ki67), cytotoxic potential (for example, granzymes and perforin) and cytokine receptors (CD122 and CD127). There is an interesting correlation between pre-therapy levels of chemokine IP-10 and response against PEG IFN-gamma / rivabirin. IP-10 levels were measured to investigate a possible correlation between negative regulatory pathways or HCV-specific T lymphocyte responses and IP-10 levels. Expression in receptors and ligands in PBMC was performed by flow cytometry.

Example 9: Enhancement of specific immunity of SIV in vivo by blocking PD-1

The potential for immune restoration of PD-1 blockade during chronic simian immunodeficiency virus (SIV) infection was tested in macaques. Fourteen rhesus macaques from India (Macaca mulatta) infected with SIV were studied. Eight macaques were used for the chronic early phase and infected intravenously with an infectious dose of 50% tissue culture of 200 (DICT50) of SIV251. Six macaques were used for the late chronic phase, three were infected with SIV 251 intrarectally and three were infected with SIV239 intravenously. All macaques, except RDb11, were negative for the Mamu B08 and Mamu B17 alleles. RDb11 was positive for the Mamu B17 allele.

In vivo antibody treatment: the macaques were infused with the partially humanized mouse anti-human PD-1 antibody (clone EH12-1540) (Dorfman et al., Am. J. Surg. Pathol. 30: 802-810, 2006) or a control antibody (SYNAGIS). The anti-PD-1 antibody has a variable mouse heavy chain domain linked to human IgG 1 (mutated to reduce the binding of FcR and complement) and a mouse variable chain domain linked to human k. Clone EH12 binds to macaque PD-1 and blocks interactions between PD-1 and its ligands in vitro. SYNAGIS is a humanized mouse monoclonal antibody (IgG1K) specific against the respiratory syncytial virus protein F. Antibodies were administered intravenously to 3 mg kg-1 body weight on days 0, 3, 7 and 10.

Immune responses: peripheral blood mononuclear cells and lymphocytes from rectal function biopsies were isolated as described previously (Velu et al., J. Virol. 81: 5819-5828, 2007). Tetramer staining, intracellular cytokine production and anti-SIV Env binding antibody measurements were performed as described above (Amara et al, Science 292: 69-74, 2001; Kannanganat et al., J. Virol 81: 84688476, 2007; Lai et al., Virology 369: 153-167, 2007).

The PD-1 block was performed during the early phase (10 weeks) as well as the late phase (approximately 90 weeks) of chronic SIV infection. Nine macaques (five during the early phase and four during the late phase) received the anti-PD-1 antibody and five macaques (three during the early phase and two during the late phase) received an isotype control antibody (SYNAGIS, specific against respiratory syncytial virus (RSV).

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Blocking PD-1 during chronic SIV infection resulted in rapid expansion of SIV-specific CD8 T lymphocytes in the blood of all macaques. The responses of CD8 T cells against two immunodominant epitopes, Gag CM9 (Allen et al., J. Immunol. 160: 6062-6071, 1998) and Tat SL8 / TL8 (Allen et al., Nature 407: 386-390, 2000 ), were studied using tetrameric complexes of the major histocompatibility complex (MHC) I in seven of the macaques treated with anti-PD-1 antibody and three of the macaques treated with control antibody expressing the Mamu A * 01 histocompatibility molecule. Most of the Gag-CM9 tetramer specific CD8 T lymphocytes (> 98%) expressed PD-1 before blockage. After the PD-1 blockade, Gag-CM9 tetramer specific CD8 T lymphocytes expanded rapidly and reached a peak after 7-21 days. In the peak response, these levels were approximately 2.5 to 11 times higher than their respective levels on day 0 (P = 0.007) and remained elevated until days 28-45. Similar results were observed with blockage during the early as well as late stages of chronic SIV infection. There was also a 3-4 fold increase in the frequency of interferon-positive CD8 T-lymphocytes (IFN) - and Gag-specific on day 14 after blockade in two animals negative to Mamu A * 01 (RTd11 and RDb11), demonstrating that blocking PD-1 can enhance the frequency of virus-specific CD8 T lymphocytes that are restricted by non-Mamu A * 01 alleles. The expansion of SIV-specific CD8 T lymphocytes was not observed in macaques treated with control antibody.

PD-1 blockade was also associated with a significant increase in the frequency of virus-specific CD8 T lymphocytes that underwent active cell division in vivo with improved functional quality. Consistent with the rapid expansion of SIV-specific CD8 T lymphocytes, the frequency of Gag-CM9 tetramer specific CD8 lymphocytes co-expressing Ki67 (marker for proliferative cells) also increased as early as day 7 after blockage (P = 0, 01). Similarly, an increase in the frequencies of Gag-CM9 tetramer specific CD8 T lymphocytes coexpressing perforin and granzyme B (cytolytic potential; P = 0.001 and P = 0.03, respectively), CD28 (costimulation potential; P = 0.001), CD127 (proliferative potential; P = 0.0003) and CCR7 (lymph node search potential; 0.001). A transient increase of 1.5 to 2 times in the frequency of CD8 T lymphocytes negative to tetramer and Ki67 positive after blockade was observed. This could be due to the expansion of specific CD8 T lymphocytes against other epitopes in Gag, as well as against other SIV proteins and other chronic viral infections in these animals. No significant potentiation was observed for these markers in the three macaques treated with control antibody.

No expansion was observed for Tat-TL8 specific CD8 T lymphocytes after blockage. This could be due to the viral escape of the recognition by Tat-TL8-specific CD8 T lymphocytes, since it is known that PD-1 blockade produces T-lymphocyte expansion only when they simultaneously receive signals through the T-lymphocyte receptor. possibility, the viral genomes present in the plasma just before the start of the blockade of the three Mamu A * 01 positive macaques that were infected with SIV 251 and received the blocking antibody during the early infection phase were sequenced. In fact, mutations were found in the viral genome corresponding to the Tat TL8 epitope region. All these mutations have been observed or predicted to reduce the binding of the Tat SL8 / TL8 peptide to the MHC Mamu A * 01 molecule and produce an escape from recognition by specific CD8 T-lymphocytes of Tat-SL8 / TL8. These results suggest that PD-1 blockade in vivo may not result in the expansion of T lymphocytes that are specific for viral epitope escape mutants.

The PD-1 blockage also results in the expansion of Gag-CM9-specific CD8 T lymphocytes in the colorectal mucous tissue (intestine), a preferential site of SIV / HIV replication. No expansion was observed in two of the seven macaques, although the expansion was obvious to one of them in blood. Unlike the blood, the intestine increased much later on day 42 and varied 2 to 3 times compared to its levels on the respective day 0 (P = 0.003). Similar to blood, Gag-CM9 tetramer specific cells co-expressing Ki67 (P = 0.01), perforin (P = 0.03), granzyme B (P = 0.01) and CD28 (P = 0.01 ) also increased in the intestine after blockage.

Most importantly, PD-1 blockade also enhanced the functional quality of antiviral CD8 T lymphocytes and resulted in the generation of polyfunctional cells capable of jointly producing IFN-y cytokines, tumor necrosis factor (TNF) - ae interleukin (IL-2). On the day of the start of the PD-1 block during the late chronic phase of infection, the frequency of IFN-positive and Gag-specific cells was also low and they failed to coexpress TNF-a and IL-2. However, after blocking, the frequency of IFN-positive cells and increase in the four macaques treated with PD-1 antibody (P = 0.03) and acquired the ability to coexpress TNF-a and IL-2. The expansion of IFN-positive cells and increased at 14-21 days and the maximum levels were 2-10 times higher than the respective day or 0 levels. On day 21, approximately 16% of the total Gag specific cells co-expressed the 3 cytokines, and approximately 30% coexpressed IFN-y and TNF-a. This differs from <1% of the total Gag specific cells that coexpress the three cytokines (P = 0.01) and approximately 14% that coexpress IFN-y and TNF-at day 0 (P = 0.04). Similar results were also observed after blockage during the early chronic infection phase.

To test the function of PD-1 in the regulation of the function of B lymphocytes during infections by the chronic immunodeficiency virus, the B lymphocyte responses after PD-1 blockade in SIV infected macaques were characterized. Analysis of PD-1 expression in subsets of different B cells before blocking with PD-1 revealed preferential expression of PD-1 by memory B cells (CD20 + CD27 + CD21-) in

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comparison with untreated B lymphocytes (CD20 + CD27-CD21 +; P <0.001). In vivo blockade of PD-1 produced a 2 to 8 fold increase in the titre of binding antibodies specific to SIV on day 28 after blockade (P <0.001). To understand this further, experiments were carried out against the proliferation of memory B lymphocytes in SIV-infected macaques that were treated simultaneously with anti-PD-1 antibody and antiretroviral therapy and a significant increase in Ki67 + memory (proliferation) was observed, but no untreated B lymphocytes as early as day 3. These results demonstrate that the PD-1-PDL pathway could have a function in the regulation of B lymphocyte alteration during chronic SIV infection.

The neutralization tests revealed a two-fold increase in titers against SIV 251 adapted to the easily neutralizable laboratory and no increase in titres against SIV 251 or SIV 239 wild-type difficult to neutralize. In two of the nine animals treated with anti-PD-1 antibody, only a minimal expansion (<2 times) of SIV specific antibody after blocking. Notably, the frequency of total memory B cells in these two animals was lower (-40% of total B cells) compared to the remaining seven animals (60-90% of total B cells) before blocking, indicating that the level of specific SIV memory B lymphocytes before blocking can determine the level of expansion of SIV specific antibodies after blocking.

PD-1 blockade produces significant reductions in plasma viremia (P = 0.03) and also prolongs the survival of SW-infected macaques (P = 0.001). In two of the five macaques treated with anti-PD-1 antibody during the early chronic phase, the viral load decreased on day 10 and persisted at or below this level until day 90. In a macaque the viral load decreased temporarily and in the other two macaques increased temporarily and returned to pre-block levels. Unlike the early chronic phase, the four macaques treated with the anti-PD-1 antibody during the late chronic phase showed a transient increase in viremia on day 7, but quickly reduced the virus load on day 21 to levels that were below their respective levels on day 0. However, viral RNA levels returned to levels prior to blockade on day 43. As expected, no significant reductions in plasma viral loads were observed in any of the 5 macaques treated with the control antibody. At 21-28 days after blocking, viral RNA levels in animals treated with anti-PD-1 antibody were 2-10 times lower than their respective day 0 levels (P = 0.03). On day 150 after the blockade, 4 of the 5 macaques in the control group died due to AIDS-related symptoms (for example, loss of appetite, diarrhea, weight loss), while the 9 animals in the antibody-treated group anti-PD-1 survived (P = 0.001).

The initial increase observed in plasma viremia levels in all animals treated in the late phase and some in the early phase could be due to an increase in the frequency of activated CD4 T lymphocytes. To determine this, the percentage of Ki67 positive total CD4 T lymphocytes was measured, as well as the frequency of IFN-producing CD4 T lymphocytes and SIV specific (preferential targets for virus replication) after blockage. These analyzes revealed a temporary increase in the percentage of CD4 T lymphocytes positive for Ki67 on days 7-14 after blockade (P = 0.002) and this increase was greater in animals treated during the late phase than in the early infection phase (P = 0.015). Similarly, an increase in the frequency of Gag-specific CD4 T lymphocytes was observed, but only in animals treated during the late infection phase. No significant increases were observed for these activated CD4 T lymphocytes in the macaques treated with control antibody. These results suggest that activated CD4 T lymphocytes could have contributed to the initial increase observed in plasma viremia levels after blockage.

After the onset of PD-1 blockade, the benchmark of viral load in plasma and total CD4 T lymphocytes in blood and intestine was similar between the groups treated with control antibody and treated with anti-PD-1 antibody. However, the frequencies of CM9 + Gag cells and CM9 + Gag cells coexpressing perforin, granzyme B or CD28 were not similar between the two treatment groups before blocking in vivo. This increases the possibility that these differences may have contributed to the expansion of CM9 + Gag cells after the PD-1 block. To study the influence of the frequency of CM9 + Gag cells before the block on their expansion after the block, the group treated with anti-PD-1 antibodies was divided into two subgroups based on the frequency of CM9 + Gag cells before the start of the block such that one group had similar levels and the other group had higher levels of CM9 + Gag cells compared to the group treated with control antibody. These subgroups were analyzed later with respect to the expansion of CM9 + Gag cells after blockage. The expansion of CM9 + Gag cells was evident in both subgroups of animals after the PD-1 block, regardless of whether they had low or high levels before the block. Similar results were also observed with subgroup analysis based on the frequency of CM9 + Gag cells that co-expressed molecules associated with a better function of T lymphocytes such as perforin, granzyme B, CCR7, CD127 or CD28. However, a trend towards a better expansion of CM9 + CD28 + Gag lymphocytes was observed in animals with higher levels of CM9 + CD28 + Gag cells before blockage, which suggests that the expression of CD28 can serve as a biomarker to predict the outcome of the blockage of PD-1 in vivo.

The experiments described above demonstrate that blocking PD-1 using an antibody against PD-1 results in rapid expansion of virus-specific CD8 T lymphocytes with improved functional quality. This enhanced T lymphocyte immunity was observed in the blood and also in the intestine, a major reservoir of SIV infection. PD-1 blockade also produced proliferation of memory B cells and increases in

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SIV envelope specific antibody. These enhanced immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. The blockade was effective during the early phase (week 10) as well as the late phase (approximately week 90) of chronic infection even in conditions of severe lymphopenia. These results demonstrate potentiation of both cellular and humoral immune responses during an infection by the pathogenic immunodeficiency virus by blocking a single inhibitory pathway and identify a new therapeutic strategy for the control of infections by the human immunodeficiency virus.

Example 10: Enhanced proliferation of SIV-specific CD8 T lymphocytes after in vitro blocking of the PD-1: PDL pathway by a humanized PD-1 antibody and a humanized PD-L1 antibody.

The effect of a humanized anti-PD-1 antibody from EH-12.2H7 and a humanized anti-PD-L1 antibody from 29E.2A3 on the proliferative capacity of Gag-specific CD8 T lymphocytes of SIV was tested in vitro. The humanized anti-PD-1 antibody has the heavy chain variable region sequence of SEQ ID NO: 28, and the light chain variable region sequence of SEQ ID NO: 32. The humanized anti-PD-L1 antibody has the heavy chain variable region sequence of SEQ ID NO: 35 and the light chain variable region sequence of SEQ ID NO: 42. The heavy chain constant region of humanized antibodies is from human IgG4 with mutation from Ser 228 to Pro (from CPSCP to CPPCP) such that the antibody forms dimeros, and the constant region of the light chain is the constant region of the human kappa light chain. The amino acid numbering for Ser 228 is in accordance with the EU numbering system. See Aalberse et al., Immunology 105: 9-19, 2002. PBMCs obtained from macaques infected with SIV (between 3 months to 1.5 years after infection) were stained with carboxyfluorescein succinimidyl diacetate ester (CFSE) and stimulated with a set of SIV Gag peptides or culture medium for 6 days in the presence or absence of a blocking antibody. At the end of the stimulation, the cells were stained for surface CD3 and CD8 and intracellular Ki-67. The cells were then acquired in a Calibur FACS and analyzed using a FlowJo software. The lymphocytes were identified based on the dispersion, then CD8 T lymphocytes (CD3 +, CD8 +) were analyzed for the quotation by Ki-67 and CFSE. Cells with low levels of CFSE, Ki-67 +, were identified as proliferative cells.

As shown in Figure 14A, in vitro blocking of the PD-1: PD-1 ligand pathway using the anti-PD-1 Ab produces a significant increase in the proliferation of SIV-specific CD8 T lymphocyte responses. In vitro blocking using the anti-PD-L1 Ab produces a moderate increase in the proliferation of CD8 T lymphocyte responses specific to SIV (Figure 14B).

Example 11: Restoration of HCV-specific T lymphocyte proliferation by intrahepatic mononuclear cells of a persistently infected chimpance.

Intrahepatic CFSE-labeled lymphocytes (2x106) were isolated from chimpanzee 1564 that had been chronically infected with HCV genotype 1a strain H77 for more than 10 years. Intrahepatic lymphocytes were co-cultured for 6 days with 4x106 PBMC with depleted CD8, irradiated autologues that were not manipulated or boosted with overlapping peptides that encompass the entire HCV polyprotein (genotype H77 strain 1a). The cells were cultured in RPMI medium supplemented with 10% L-glutamine and FCS, with and without an anti-PD-L1 blocking antibody (10 | jg / ml, added on day 0 and day 2). The humanized anti-PD-L1 antibody has the heavy chain variable region sequence of SEQ ID NO: 35 and the light chain variable region sequence of SEQ ID NO: 42. The heavy chain constant region of humanized antibodies is of human IgG4 with Ser 2228 to Pro mutation (from CPSCP to CPPCP) so that the antibody forms dimeros and the light chain constant region is the human kappa light chain constant region. The amino acid numbering for Ser 228 is in accordance with the EU enumeration system. See Aalberse et al., Immunology 105: 9-19, 2002. On day 6, the cells were stained with CD8-PerCP, tetramer A0701 / P7 (758) -PE, PD-1-Alexa 647, CD4-Alexa 700, CD14-Alexa 700, CD16-Alexa 700, CD19-Alexa 700 and Live / Dead blue dye. Samples were acquired on a BD LSR II flow clitometer and the data was analyzed using a FlowJo software.

As shown in Figure 15, treatment with the anti-PD-L1 antibody reestablished the proliferation of IICV-specific T lymphocytes by intrahepatic mononuclear cells of a persistently infected chimpance.

Claims (16)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 55 60 65 1. An isolated antibody or an antigen binding fragment thereof, comprising: a) a heavy chain variable region sequence selected from the group consisting of SEQ ID NO: 34-38; Y b) a light chain variable region sequence selected from the group consisting of SEQ ID NO: 3942, wherein the isolated antibody, or an antigen binding fragment thereof, binds to a PD-L1 protein having the amino acid sequence of SEQ ID NO: 4, and the isolated antibody, or the antigen binding fragment of it, is humanized or is composed. 2. The isolated antibody or antigen binding fragment of claim 1, wherein the isolated antibody comprises: a) a heavy chain variable region sequence comprising SEQ ID NO: 35 or 37; Y b) a light chain variable region sequence comprising SEQ ID NO: 39, 40 or 42. 3. The isolated antibody or antigen binding fragment of claim 1 or claim 2, wherein the isolated antibody or antigen binding fragment thereof inhibits the binding of antibody 29E2A3 to Fc-PD-L1, wherein antibody 29E2A3 comprises a heavy chain variable region sequence comprising SEQ ID NO: 78, and a light chain variable region sequence comprising SEQ ID NO: 79. 4. The isolated antibody or antigen binding fragment of claim 1 or claim 2, wherein the isolated antibody or antigen binding fragment thereof inhibits a signal mediated by PD-L1. 5. An isolated nucleic acid encoding the antibody or antigen binding fragment of any one of claims 1-4. 6. A vector comprising the isolated nucleic acid of claim 5. 7. A host cell comprising one or more nucleic acids encoding the heavy and light chains of the antibody or antigen binding fragment of any one of claims 1-4. 8. The host cell of claim 7 producing the antibody or antigen binding fragment encoded by the nucleic acid. 9. A transgenic non-human animal comprising one or more nucleic acids encoding the heavy and light chains of the antibody or antigen binding fragment of any one of claims 1-4. 10. A pharmaceutical composition, comprising the isolated antibody or the antigen binding fragment of any one of claims 1-4 and a pharmaceutically acceptable carrier. 11. A method of reactivation of depleted T lymphocytes, comprising: contacting a population of cells in which at least some cells express PD-L1, with an effective amount of a composition comprising an antibody, or an antigen binding fragment thereof, of any one of claims 1-4 , where the stage of contacting is performed in vitro or ex vivo. 12. An antibody, or an antigen binding fragment thereof, of any one of claims 1-4, for use in: (1) a method of treatment of a subject suffering from a persistent infection; or (2) a method of treatment of a subject suffering from a viral infection, wherein the viral infection is optionally selected from the group consisting of cytomegalovirus, Epstein-Barr virus, hepatitis B, hepatitis C virus, herpes virus, human immunodeficiency virus, human T lymphotropic virus, lymphocytic choriomeningitis virus, respiratory syncytial virus and / or rhinovirus; or (3) a method of treating a subject suffering from a bacterial infection, wherein the bacterial infection is optionally selected from the group consisting of Helicobacter, Mycobacterium, Porphyromonas and Chlamydia; or (4) a method of treatment of a subject suffering from a helminthic infection, wherein the helminth is optionally selected from the group consisting of Schistosoma and Taenia; or (5) a method of treating a subject suffering from a protozoal infection, wherein the protozoal infection is optionally selected from the group consisting of Mexican Leishmania and Plasmodium; or 10 fifteen (6) a method of treating a subject suffering from cancer, wherein the cancer is optionally selected from the group consisting of a solid tumor, a hematological cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary cancer, stomach cancer, thymic epithelial cancer and thyroid cancer; or (7) a method of treatment of a subject suffering from a cancer that overexpresses PD-L1. 13. The antibody for use of claim 12, part (7), which induces antibody-mediated cytotoxicity. 14. The antibody for use of claim 13, which is modified to increase antibody-induced cytotoxicity. 15. The antibody for use of claim 14, which is conjugated to an agent selected from the group consisting of a toxin and an imaging agent. 16. A method of producing the antibody or antigen binding fragment of any one of claims 1-4, comprising culturing a cell that produces the antibody or antigen binding fragment and recovering the antibody or cell from the cell culture. antigen binding fragment.